JP2007521331A - Methods of treating diseases and disorders by targeting multiple kinases - Google Patents

Abstract

The present invention relates to the use of a single drug that is a compound that simultaneously targets two or more kinases so as to substantially avoid resistance to therapy. The present invention relates to a disease or condition comprising administering one or more single drugs alone or in combination with other treatments to treat various diseases or conditions related to the activity of two or more kinases. Methods of use for administering to an individual and for treating the individual are provided.
[Selection] Figure 1

Description

  The present invention encompasses methods for simultaneously modulating the activity of multiple kinases or kinase pathways, including those involved in processes important for cell survival, proliferation, growth and malignant transformation, motility and invasion. Thus, the present invention includes conditions, diseases and disorders related to protein kinases or protein kinase pathways, such as proliferative diseases, cancers and inflammatory diseases, including diabetes, obesity, and abnormal angiogenesis and related diseases. Also encompassed are methods for treatment, prevention or management. In particular, the methods contemplated by the present invention include treating, preventing or managing a disease, condition or disorder with a single drug treatment that specifically targets multiple kinases or kinase pathways. In one embodiment, it is preferred that a single drug treatment modulates the activity of multiple kinases or kinase pathways over the activity of other kinases. In other words, the effects of single drug treatment are selective for a specific set of kinases. In summary, the present invention contemplates the identification and use of a single drug that targets the appropriate combination of multiple pathways that are clinically effective in a particular disease environment.

  The relationship between abnormal protein phosphorylation and disease cause or prognosis has been known for over 20 years. Protein kinases therefore represent a very important group of drug targets. See Cohen, Nature, 1: 309-315 (2002). Various protein kinase inhibitors have been used clinically in the treatment of various diseases such as cancer and chronic inflammatory diseases including diabetes and stroke. Cohen, Eur. J. et al. Biochem. 268: 5001-5010 (2001).

  Protein kinases are a large and diverse family of enzymes that catalyze protein phosphorylation and play an important role in cell signaling. Protein kinases can exert positive or negative regulatory effects depending on the target protein. Protein kinases are involved in specific signal transduction pathways that regulate cell functions, which include, but are not limited to, metabolism, cell cycle progression, cell adhesion, vascular function, apoptosis and angiogenesis. Cellular signaling dysfunction is associated with many diseases, with the most characteristic diseases including cancer and diabetes. Regulation of signal transduction by cytokines and the relationship between signal molecules and proto-oncogenes and tumor suppressor genes have also been demonstrated. The relationship between diabetes and related conditions and deregulation levels of protein kinases has also been demonstrated. See, for example, Sridhar et al., Pharmaceutical Research, 17 (1): 1345-1353 (2000). Viral infections and associated conditions are also implicated in the regulation of protein kinases. Park et al., Cell, 101 (7): 777-787 (2000).

  Protein kinases can be divided into broad groups based on the identity of the amino acids that they target (serine / threonine, tyrosine, lysine and histidine). For example, tyrosine kinases include receptor tyrosine kinases (RTK) such as growth factors and non-receptor tyrosine kinases such as the src kinase family. There are also bispecific protein kinases that target both tyrosine and serine / threonine, such as cyclin dependent kinases (CDK) and mitogen activated protein kinases (MAPK). Certain cells contain many protein kinases, some of which phosphorylate other protein kinases. Some protein kinases phosphorylate a wide variety of proteins, others phosphorylate only one protein. As expected, there are many classes of protein kinases. Upon receiving a signal, some proteins may undergo autophosphorylation.

  Protein tyrosine kinases (PTKs) constitute a large family of kinases that regulate cells to cellular signals involved in proliferation, differentiation, adhesion, motility and death. Robinson et al., Oncogene, 19: 5548-5557 (2000). Tyrosine kinase members include, but are not limited to, Yes, BMX, Syk, EphA1, FGFR3, RKY, MUSK, JAK1 and FGFR. Tyrosine kinases are divided into two classes: receptor tyrosine kinases and non-receptor tyrosine kinases. Interestingly, the entire tyrosine kinase family is very large, consisting of at least 90 characterized kinases of at least 58 receptor kinases and at least 32 non-receptor kinases, totaling at least 30 subfamilies. including. Robinson et al., Oncogene, 19: 5548-5557 (2000). Tyrosine kinases are involved in a number of diseases, including diabetes and cancer in humans. Robinson et al., P. 5548. Tyrosine kinases are often implicated in many forms of human malignancy and have been linked to various congenital syndromes. Robinson et al., Trends Genet. 16: 265-271 (2000).

  Non-receptor tyrosine kinases are a group of intracellular enzymes that do not contain extracellular and transmembrane sequences. Currently, over 32 non-receptor tyrosine kinase families have been identified. Robinson et al., Oncogene, 19: 5548-5557 (2000). For example, Src, Btk, Csk, ZAP70, Kak family. In particular, the Src family of non-receptor tyrosine kinase families is the largest and is composed of Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk protein tyrosine kinases. The Src family of kinases has been linked to carcinogenesis, cell proliferation and tumor progression. Non-receptor protein tyrosine kinases are discussed in detail in Oncogene, 8: 2025-2031 (1993). Many of these protein tyrosine kinases have been shown to be involved in cell signaling pathways involved in various pathological conditions. Said pathological conditions include, but are not limited to, cancer, hyperproliferative diseases and immune diseases.

The cyclin-dependent kinase CDK is a group of intracellular enzymes that control progression through the cell cycle and have an essential role in cell proliferation. See Cohen, Nature, 1: 309-315 (2002). Examples of CDKs include, but are not limited to, cyclin dependent kinase 2 (CKD2), cyclin dependent kinase 7 (CDK7), cyclin dependent kinase 6 (CDK6) and cell division control 2 protein (CDC2). CDK regulates the transition between different phases of the cell cycle, eg from the resting phase of G ₁ (gap between mitosis and initiation of DNA replication for a new round of cell division) to S (period of active DNA synthesis) are involved in the progression to active mitosis and cell division occurs M phase from the progression or G _2. For example, articles can be found in Science, 274: 1643-1667 (1996) and Ann. Rev. Cell Dev. Biol. 13: 261-291 (1997). The CDK complex is an association of regulatory cyclin subunits (eg, cyclins A, B1, B2, D1, D2, D3 and E) and catalytic kinase subunits (eg, cdc2 (CDK1), CDK2, CDK4, CDK5 and CDK6). It is formed by. As the name implies, CDKs show absolute dependence on cyclin subunits to phosphorylate target substrates and function to regulate the progression of different kinase / cyclin pairs through specific parts of the cell cycle. To do. CDK is involved in various pathological conditions, which include, but are not limited to, diseases exhibiting a cancer phenotype, various neoplastic diseases and neurological diseases. Hunter, Cell, 100: 113-127 (2000).

  Mitogen-activated protein (MAP) kinases are involved in transmitting signals to the cell nucleus in response to extracellular stimuli. Examples of MAP kinases include mitogen activated protein kinase 3 (MAPK3), mitogen activated protein kinase 1 (EPK2), mitogen activated protein kinase 7 (MAPK7), mitogen activated protein kinase 8 (JNK1), mitogen activated protein Examples include, but are not limited to, kinase 14 (p38 alpha), mitogen activated protein kinase 10 (MAPK10), JNK alpha protein kinase, stress activated protein kinase JNK2 and mitogen activated protein kinase 14 (MAPK14). MAP kinases are a family of proline-directed serine / threonine kinases that mediate signal transduction from extracellular receptors or heat shock or UV radiation. See Sridhar et al., Pharmaceutical Research, 17 (11): 1345-1353 (2000). MAP kinase is activated by phosphorylation of threonine and tyrosine by bispecific protein kinases including tyrosine kinases such as growth factors. Cell proliferation and differentiation has been found to be under regulatory control of multiple MAP kinase cascades. See Sridhar et al., Pharmaceutical Research, 17 (11): 1345-1353 (2000). Thus, the MAP kinase pathway plays an important role in many pathological conditions. For example, it has been found that insufficient MAP kinase activity results in abnormal cell proliferation and carcinogenesis. Hu et al., Cell Growth Differ. 11: 191-200 (2000) and Das et al., Breast Cancer Res. Treat. 40: 141 (1996). Furthermore, MAP kinase activity is also involved in insulin resistance associated with type 2 diabetes. Virkamaki et al., J. MoI. Clin. Invest. 103: 931-943 (1999).

  p90 ribosomal S6 kinase (Rsk) is a serine / threonine kinase. Rsk family members function in mitogen-activated cell growth and proliferation, differentiation and cell survival. Examples of members of the Rsk family of kinases include ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (Rsk3), ribosomal protein S6 kinase, 90 kDa, polypeptide 6 (Rsk4), ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (Rsk2 ) And ribosomal protein S6 kinase, 90 kDa, polypeptide (Rsk1 / p90Rsk). Rsk family members are activated by extracellular signal-related kinase 1/2 and phosphoinositide-dependent protein kinase 1. Frodin and Gameltoft, Mol. Cell. Endocrinol. 151: 65-77 (1999). Under basal conditions, RSK kinases are localized in the cytoplasm of the cell, and when stimulated by mitogens, activated RSK (phosphorylated by extracellular associated kinases) temporarily translocates to the plasma membrane where it is completely Activated. Fully activated RSK phosphorylates substrates involved in cell growth, proliferation, differentiation and cell survival. Richards et al., Curr. Biol. 9: 810-820 (1999); Richards et al., Mol. Cell. Biol. 21: 7470-7480 (2001). The RSK signaling pathway is also involved in cell cycle modulation. Gross et al. Biol. Chem. 276 (49): 46099-46103 (2001). Current data suggests that small molecules that inhibit Rsk may be useful therapeutic agents for preventing and treating cancer and inflammatory diseases.

  Members of the checkpoint protein kinase family are serine / threonine kinases that play an important role in cell cycle progression. Examples of checkpoint family members include, but are not limited to, CHK1 and CHK2. Checkpoints are control systems that synchronize cell cycle progression by affecting the formation, activation and subsequent inactivation of cyclin-dependent kinases. Checkpoints prevent cell cycle progression when inappropriate, maintain cell metabolic balance while cells are stopped, and sometimes apoptotic (programmed cell death) when checkpoint requirements are not met Can be induced. See, for example, O'Connor, Cancer Surveys, 29: 151-182 (1997); Nurse, Cell, 91: 865-867 (1997); Hartwell et al., Science, 266: 1821-1828 (1994); Hartwell et al., Science, 246: 629-634 (1989). Members of the checkpoint family of kinases are involved in cell proliferative disorders, cancer phenotypes, and other diseases associated with DNA damage and repair. Kohn, Mol. Biol. Cell, 10: 2703-2734 (1999); Ohi and Gould, Curr, Opin. Cell Biol. 11: 267-273 (1999); Peng et al., Science, 277: 1501-1505 (1997).

  Aurora kinases are a family of multigene mitotic serine-threonine kinases that function as a class of novel oncogenes. These kinases include Aurora A and Aurora B members. Aurora kinase is over-activated and / or over-expressed in some solid tumors. Said solid tumors include but are not limited to breast cancer, ovarian cancer, prostate cancer, pancreatic cancer and colorectal cancer. In particular, Aurora A is a centrosome kinase that plays an important role in cell cycle progression and cell proliferation. Aurora A is located on chromosome 20q13, which is frequently grown in several different types of malignancies such as colorectal cancer, breast cancer and bladder cancer. There is also a high correlation between Aurora A and high tissue prognostic grade aneuploidy, making kinase a potential prognostic mediator. Inhibition of Aurora kinase activity can help reduce cell proliferation, tumor growth and potentially tumor formation. A detailed description of Aurora kinase function is discussed in Oncogene, 21: 6175-6183 (2002).

  Rho-related coiled-coil-containing proteins serine / threonine kinases ROCK-I and ROCK-II play an important role in cytoskeletal dynamics by acting as downstream effectors of the Rho / Rac family of cytokine- and growth factor-activated small GTPases it is conceivable that. ROCK phosphorylates various substrates. Such substrates include, but are not limited to, myosin light chain phosphatase, myosin light chain, ezrin-radixin-moesin protein and LIM (relative to Lin11, Isl1 and Mec3) kinases. ROCK also mediates actin stress fiber formation and focal adhesion in various cell types. ROCK has an important role in cell migration by enhancing cell contractility. ROCK requires monocyte and cancer cell tail contraction, and ROCK inhibitors have been used to reduce tumor cell seeding in vivo. Recent experiments have defined new functions of ROCK in cells including centrosome positioning and cell size regulation that can contribute to various physiological and pathological conditions. Nature Reviews Mol. Cell Biol. 4: 446-456 (2003). ROCK family members are attractive intervention targets for a variety of conditions, including cancer and cardiovascular disease. For example, Rho kinase inhibitors can be useful therapeutics for hypertension, angina and asthma. Furthermore, Rho is expected to play a role in peripheral circulatory diseases, atherosclerosis, inflammation and autoimmune diseases and is therefore a useful target for treatment.

  The 70 kDa ribosomal S6 kinase (p70S6K) is activated by multiple mitogens, growth factors and hormones. Activation of p70S6K occurs by phosphorylation at multiple sites, and the primary target of activated kinase is 40S ribosomal protein S6, which is a major component of the mechanism involved in protein synthesis in mammalian cells. In addition to being involved in the regulation of translation, p70S6K activation is involved in cell responses that are important in cell cycle control, neuronal cell differentiation, regulation of cell motility, and tumor metastasis, immunity and tissue repair. Modulation of p70S6K kinase activity may be therapeutically relevant in diseases such as cancer, inflammation and various neurological disorders. p70S6K kinase is described in Prog. Cell Cycle Res. 1: 21-32 (1995) and Immunol. Cell Biol. 78 (4): 447-51 (2000).

  Glycogen synthase kinase 3 (GSK-3) phosphorylates cellular substrates and thus regulates a wide variety of cellular functions including development, metabolism, gene transcription, protein translation, cytoskeletal organization, cell cycle regulation and apoptosis A constitutively active serine / threonine kinase that is ubiquitously expressed. GSK-3 was first described as the major enzyme involved in glycogen metabolism, but is now known to regulate various cellular functions. Two forms of enzyme have already been identified, namely GSK-3α and GSK-3β. The activity of GSK-3β is negatively regulated by protein kinase B / Akt and Wnt signaling pathways. Thus, small molecule inhibitors of GSK-3 have several therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes, bipolar disorder, stroke, cancer and chronic inflammatory diseases. The role of glycogen synthase kinase 3 in cancer is regulated by Wuts and other signaling pathways (Adv. Cancer Res., 84: 203-29 (2002)); new promising for diabetes, neurodegeneration, cancer and inflammation Glycogen synthase kinase 3 (GSK-3) inhibitor as a drug (Med. Res. Rev., 22 (4): 373-84 (2002)); glycogen synthase kinase 3 in the phosphatidylinositol 3-kinase / Akt cell survival pathway (J. Biol. Chem., 273 (32): 19929-32 (1998)).

  Protein kinases are attractive targets for the treatment of various pathological conditions because they regulate almost all cellular processes, including metabolism, cell proliferation, cell differentiation and cell survival. For example, cell cycle control and angiogenesis, where protein kinases play an important role, are cellular processes that are associated with a number of pathological conditions. Such pathological conditions include, but are not limited to, cancer, inflammatory diseases, abnormal angiogenesis and related diseases, atherosclerosis, macular degeneration, diabetes, obesity and pain.

  Protein kinases have become attractive targets for cancer treatment. Fabbro et al., Pharmacology & Therapeutics, 93: 79-98 (2002). The involvement of protein kinases in the development of human malignancies is constitutively active kinase activity such as (1) genome transposition (eg BCR-ABL in chronic myelogenous leukemia), (2) acute myeloid leukemia and gastrointestinal tumors (3) deregulation of kinase activity due to oncogene activation or loss of tumor suppressor function, as in the case of cancer with the oncogene RAS, (4) kinase by overexpression as in EGFR It has been proposed that it can be caused by deregulation of activity and (5) ectopic expression of growth factors that can contribute to the development and maintenance of a neoplastic phenotype. Fabbro et al., Pharmacology & Therapeutics, 93: 79-98 (2002).

  Certain cancers are associated with angiogenesis. Angiogenesis is the growth of new capillaries from existing vasculature. W. Risau, Nature, 386: 671-674 (1997). It has been found that protein kinases can contribute to the development and maintenance of a neoplastic phenotype. Fabbro et al., Pharmacology & Therapeutics, 93: 79-98 (2002). For example, VEGF AD and its four receptors are involved in phenotypes including neovascularization and high vascular permeability such as tumor angiogenesis and lymphangiogenesis. A. Matter, Drug Discov. Today, 6: 1005-1023 (2001).

  Cardiovascular disease (CVD) accounts for almost one quarter of all deaths worldwide. Vascular disorders such as atherosclerosis and restenosis occur due to dysregulated growth of the vessel wall and restriction of blood flow to vital organs. Various kinase pathways (eg, JNK) are activated by atherogenic stimuli and are regulated by local cytokine and growth factor production in vascular cells. Yang et al., Immunity, 9: 575 (1998). Ischemia and ischemia with reperfusion in the heart, kidney or brain can result in cell death and scar formation, which can ultimately lead to congestive heart failure, renal failure or brain dysfunction. In the case of organ transplantation, reperfusion of the ischemic donor organ already results in acute lymphocyte-mediated tissue damage and delayed graft function. Ischemic and reperfusion pathways are mediated by various kinases. For example, the JNK pathway is linked to leukocyte-mediated tissue damage. Li et al., Mol. Cell. Biol. 16: 5947-5954 (1996). Finally, high apoptosis in heart tissue is also linked to kinase activity. Pombo et al. Biol. Chem. 269: 26546-26551 (1994).

  Protein kinase modulators, modulators or inhibitors whose elucidation of the complexity of protein kinase pathways and the relationships and interactions between various protein kinases and kinase pathways has advantageous activity against multiple kinases or multiple kinase pathways Stresses the importance of developing pharmaceuticals that can act as

  It has been recognized that a single drug approach that specifically targets one kinase or one kinase pathway may be insufficient for several reasons to treat diseases and disorders, particularly cancer. The single drug approach is a limited approach to treating very complex diseases, conditions and disorders. For example, mathematical models suggest that 5-7 mutations are required for progression from normal cells to malignant transformation. In addition, other events that modify the expression of existing genes can occur (eg, DNA methylation).

  Thus, it is widely accepted that cancer is the result of changes in protein kinase pathways associated with multiple pathways, particularly processes such as cell growth, proliferation, apoptosis, motility or invasion. A common requirement in many cancers is the simultaneous overexpression and / or overactivation of various protein kinases (eg, receptor and non-receptor kinases, serine / threonine kinases, P13 kinases and cell cycle related kinases). Indeed, some of these kinases alone or together with other kinases, numerous processes and diseases important for cell survival, proliferation, growth and malignant transformation, motility and invasion leading to metastasis and angiogenesis or inflammation, Involved in faults and related conditions.

  Thus, since there are multiple target kinases that affect the progression of a condition, disease or disorder, it may not be clinically sufficient to block one target kinase alone. In addition, blocking a single target kinase may not be clinically sufficient because abundant kinase-mediated pathways and other oncogenic or inflammatory mechanisms can compensate for the blocked target kinase. In addition, the use of a single drug may increase the likelihood of developing resistance to the drug.

  Thus, due to the complexity of the intracellular signaling cascade of the protein kinase pathway, it has been suggested that drugs that simultaneously affect multiple pathways may be required for significant clinical activity. Although it has been suggested that a single drug that provides a combined effect is attractive, the identification and use of a single drug that targets the appropriate combination of multiple pathways that are clinically effective in a particular disease setting is required. is there. In fact, multiple kinase drugs such as Gleevec® are known to target several kinases at once. Gleevec® primarily targets mutant fusion proteins containing abl kinase created by the 9:22 chromosomal translocation event. Gleevec® also targets c-kit, a tyrosine kinase involved in gastrointestinal stromal tumors (GIST). However, recent clinical trials have shown that patients are resistant to Gleevec® and respond incompletely to treatment.

  It has also been suggested that a single drug can target the main cause of the disease and thereby regulate downstream pathways by targeting only one specific molecule. For example, it has been found that viral infections often affect the regulation of multiple kinases, so that drugs that inhibit viral replication can inhibit all activation of multiple kinases activated by the virus. However, due to the complexity of the intracellular signaling cascades of the protein kinase pathway and the diseases in which they are involved, a single drug that affects multiple pathways simultaneously by directly targeting specific kinases and / or kinase pathways Is needed. In particular, the identification of a single drug that can target multiple kinases or kinase pathways simultaneously and can selectively target specific kinases and / or kinase pathways directly without affecting other kinases and / or pathways. is necessary. For example, rather than using a single drug that suppresses all activations of virus-activated multiple kinases in a viral infection, a single drug selectively inhibits specific activated multiple kinases simultaneously. Can do.

  Additional strategies to target multiple kinases or kinase pathways have used single target drug cocktails. Targeted to multiple kinases or kinase pathways because the use of a combination of drugs proved promising in the clinic, but the use of a single drug is more practical for multiple reasons such as ease of administration and simplicity There is still a need for the development of single drugs that can be Furthermore, it is already known that certain treatment regimens using multiple drugs, each targeting a single pathway, can be associated with complications including undesirable side effects and toxicity.

  Accordingly, there remains a need for the development of methods that involve the use of a single drug that can target a specific set of kinases or kinase pathways, particularly an appropriate combination of multiple targets to obtain a clinical effect.

  The present invention is based in part on the finding that small molecule kinase inhibitors that can simultaneously inhibit multiple kinases (also referred to herein as mixed kinases) have stronger antiproliferative activity than certain kinase inhibitors. Is based on. Since inhibition of one specific kinase or a specific pathway is not necessarily sufficient to elicit a significant clinical response and may result in rapid resistance, the present invention provides a single target that can target more than one kinase or kinase pathway. Includes methods involving the use of one drug. Thus, the method of the present invention affects processes important for cell survival, proliferation, growth and malignant transformation, motility and invasion leading to angiogenesis and metastasis by simultaneously targeting multiple protein kinases or protein kinase pathways. It consists of an effect method.

  Thus, the present invention can target a specific kinase, ie, modulate or modulate the activity, function or level of a specific kinase and / or the expression level of a gene encoding the specific kinase; It relates to the use of drugs that modulate or modulate the pathway, ie can target kinases or kinase-mediated pathways. Said drugs are called “single drugs” or “mixed kinase drugs”.

  The Applicant has found that CDK kinase, Rsk kinase, checkpoint kinase, MAPK kinase, Src kinase and kinases Fes, Lyn, Syk are important and particularly important for the worsening of proliferative diseases. Thus, in one embodiment, the drug is a kinase from the src kinase family, a kinase from the Rsk kinase family, a kinase from the CDK family, a kinase from the MAPK kinase family and a tyrosine kinase (eg, Fes, Lyn and Syk kinase). Target more than one. The drugs can target two or more kinases of the same family, or can target two or more kinase families or classes of kinases.

  In a preferred embodiment, the present invention provides a method for treating a disease, disorder or condition as described above comprising targeting a plurality of kinases or kinase pathways involved in the disease, disorder or condition.

  The invention also encompasses combinations of a single drug with a single kinase or one or more drugs that specifically target the kinase pathway. In other embodiments, the single drug may be used for other treatments such as conventional forms of chemotherapy, anti-angiogenesis, nucleoside analogs, proteosome inhibitors, etc .; radiation therapy; cytokine therapy; surgery; or Section 4.3 below. Can be used in conjunction with other treatments described in.

  Thus, the method of the present invention is also useful as an adjunct to existing and / or experimental treatments.

  According to the methods of the invention, the identification of modulators that can simultaneously target, regulate or inhibit multiple kinases or kinase pathways critically involved in various diseases or disorders is disclosed in Examples 4-83. This can be accomplished by using the assay described or the in vivo model described in Example 3.

Definitions As used herein, “resistant” and “resistant” refer to a target kinase that exhibits a detectably reduced response to a particular kinase modulator. For example, a resistant kinase is one that has a detectable higher activity relative to the kinase response when the kinase exposed after the first contact with the single drug of the present invention is first exposed to the single drug. is there. By way of example, the kinase may be resistant to a single inhibitor or mixture of inhibitors, or may exhibit a possible reduction in response. Resistance to a particular modulator of the kinase can arise from mutations, changes in post-translational modifications, up- or down-regulation of the gene encoding the kinase, high or low clearance of the kinase from the tissue or other causes. Resistance can also arise as a result of up-regulation of alternative kinase-mediated pathways whose function is at least partially redundant to the targeted kinase pathway.

  As used herein, the terms “simultaneous” and “simultaneously” mean a specific administration and duration of effect of a single drug when a single drug is administered to an individual, and treatment is applied to all targeted kinases. It does not matter whether this effect is verifiable at the exact same specific point between administration and effect. Certain single drugs, combinations of single drugs, or combinations of one or more single drugs and one or more other compounds simultaneously target two or more kinases during therapy. For example, a single drug can be administered to an individual and the effect on one targeted kinase can be detected immediately (eg, within the first few minutes of administration) and the effect on a second targeted kinase can be detected later. In some cases, a single drug is said to affect two kinases simultaneously. “Simultaneous” and “simultaneously” also refer to similar in vitro effects that occur upon contact with a single drug.

  As used herein, a “side effect” is a modulation of the activity of two or more targeted kinases of a particular single drug, a single drug combination or a combination of a single drug and another treatment. Refers to actions other than direct effects that rate (eg, inhibit).

  As used herein, a single drug interacts with each kinase to modify, inhibit or modulate kinase activity, for example, competitively, non-competitively or uncompetitively inhibit the kinase; Interact with specific transcription complexes to alter kinase levels in tissues; alter post-translational modifications of specific kinases; etc. “directly” or “specific” for two or more target kinases Acting on. For example, whether a compound directly targets more than one kinase can be determined using the in vitro assays described herein, or using other kinase assays known in the art. Can do. It does not act “directly” on the target kinase if the effect on the target kinase is simply the result of the action of a single drug on the kinase that modifies the target kinase.

  As used herein, “therapeutically effective amount” refers to an amount of a therapeutic agent sufficient to detectably improve one or more symptoms of a disease. In certain embodiments, “therapeutically effective amount” refers to the amount of therapeutic agent sufficient to destroy, modify, control or remove primary, localized or metastatic cancer tissue. Thus, a therapeutically effective amount refers to the amount of therapeutic agent sufficient to delay or minimize the spread of cancer. In another example, a “therapeutically effective amount” refers to an amount effective to detectably suppress inflammation, production of cytokines or proliferation of cells with inflammation. A therapeutically effective amount provides a therapeutic effect in the treatment or management of a disease, condition or disorder in an individual population; or an amount of a therapeutic agent that reduces the frequency of occurrence or recurrence of one or more symptoms of a disease, condition or disorder. May point.

  As used herein, a “prophylactically effective amount” refers to an amount of a prophylactic agent sufficient to prevent the recurrence or onset of one or more symptoms of a disease, condition or disorder.

  In certain embodiments, a “prophylactically effective amount” refers to an amount of a prophylactic agent sufficient to prevent cancer recurrence or spread. Thus, this term refers to the amount of prophylactic agent sufficient to prevent recurrence or spread of cancer or the occurrence of cancer in an individual, said individual being susceptible to cancer or already exposed to a carcinogenic drug. Is included, but is not limited thereto. A prophylactically effective amount may refer to the amount of a prophylactic agent that provides a prophylactic effect in the prevention of a disease, condition, or disorder.

  As used herein, a “single drug” simultaneously targets two or more kinases or kinase pathways and is used in the prevention, treatment, management or amelioration of one or more symptoms of a disease, condition or disorder Refers to a therapeutic or prophylactic agent that can be performed. Preferably, a single drug is not a macromolecule (eg, protein, polypeptide, polysaccharide, polynucleotide), but is preferably a small organic molecule having a molecular weight of less than 1000 daltons. A single drug can be a peptide or a polynucleotide fragment (eg, an aptamer). Preferably, a single drug is orally bioavailable and / or bioactive, eg bioactive and bioavailable when delivered intramuscularly, intravenously or by inhalation. The term “single drug” does not include natural proteins that exist in native form.

  As used herein, the term “drug”, “therapeutic agent” or “prophylactic agent” targets a kinase, ie, modulates, modulates, enhances, inhibits the function, activity or expression of a protein kinase, Refers to any drug that inhibits, suppresses or neutralizes, eg, protein, polypeptide, peptide, antibody, antibody fragment, oligonucleotide, antisense oligonucleotide, large molecule or small molecule (less than 10 kD).

  As used herein, the phrase “non-responsive / refractory” is treated with the currently available treatment, but this treatment is not clinically sufficient to improve the patient's symptoms, This patient is thus used to refer to a patient who must receive additional effective treatment (eg, still insensitive to treatment). This phrase can also refer to a patient who has responded to treatment still suffering from side effects, recurrence, tolerance, etc. In the context of anti-cancer therapy, in a specific embodiment, “non-responsive / non-responsive” means that at least some significant portion of the cancer cells have not been killed and cell differentiation has stopped. Whether a cancer cell is “non-responsive / refractory” is known in the art for assessing the effectiveness of treatment against cancer cells using the art recognized meaning of “refractory”. The method can be examined in vitro or in vivo.

  In various embodiments, the cancer is “non-responsive / refractory” in which the number of cancer cells is not significantly reduced or increased.

  As used herein, the phrase “low tolerance” refers to the extent to which a patient is not effective for a patient from a condition suffering from treatment and / or cannot continue treatment due to side effects.

  As used herein, the terms “individual”, “subject” and “patient” are used interchangeably. As used herein, preferred subjects are mammals, such as non-primates (eg, cows, pigs, horses, cats, dogs, rats, etc.) or primates (eg, monkeys and humans).

  As used herein, the terms “manage”, “managing” and “management” refer to an advantageous effect in which a subject benefits from a prophylactic or therapeutic agent but does not cure the disease. In certain embodiments, a subject is administered one or more prophylactic or therapeutic agents to “manage” the disease and prevent progression or worsening of the disease.

  As used herein, the terms “prevent”, “preventing” and “prevention” refer to the prevention of the recurrence or onset of one or more symptoms of a disease, disorder or condition. In one embodiment, the term refers to preventing the recurrence or onset of one or more symptoms of an autoimmune or inflammatory disease that occurs upon administration of a prophylactic or therapeutic agent in a subject. In another embodiment, the terms “prevent”, “preventing” and “prevention” also refer to prevention of recurrence, spread or development of cancer in a subject caused by administration of a prophylactic or therapeutic agent.

  As used herein, the terms “treat”, “treatment” and “treating” refer to one or more associated with the disease, disorder or condition resulting from the administration of one or more prophylactic or therapeutic agents. Refers to improvement of symptoms. In certain embodiments, the term refers to a reduction in the proliferative activity of cells that occurs upon administration of one or more prophylactic or therapeutic agents to a subject in need of treatment. In other embodiments, the term refers to the reduction in cellular inflammatory activity that occurs upon administration of one or more prophylactic or therapeutic agents to a subject in need of treatment. In other specific embodiments, the term refers to the reduction in abnormal angiogenic activity of cells that occurs upon administration of one or more prophylactic or therapeutic agents to a subject in need of treatment.

  As used herein in connection with anti-cancer treatments, the terms “treat”, “treating” and “treatment” are primary, localized resulting from the administration of one or more prophylactic or therapeutic agents. Refers to eradication, removal, modification, reduced spread or reduced spread rate, or control of sex or metastatic cancer tissue. In certain embodiments, the term refers to minimizing or delaying the spread of cancer that occurs when one or more prophylactic or therapeutic agents are administered to a cancer patient.

  The invention encompasses methods for the treatment, prevention or management of conditions, diseases or disorders involving more than one protein kinase or protein kinase pathway. The present invention is based on the finding that small molecule kinase inhibitors that can simultaneously inhibit multiple kinases (also referred to herein as mixed kinases) have more potent anti-proliferative activity than certain kinase inhibitors. Based in part. We have two or more of kinases from the src kinase family, kinases from the Rsk kinase family, kinases from the CDK family, kinases from the MAPK kinase family and tyrosine kinases (eg, Fes, Lyn and Syk kinases). We have found ways to simultaneously modulate processes involved in cell survival, proliferation, growth and malignant transformation, motility and invasion leading to angiogenesis and metastasis by simultaneously modulating the protein kinase pathways involved. Specifically, the inventors have discovered a single drug that can target multiple kinases and / or kinase pathways simultaneously.

  The present invention targets more than one kinase or kinase pathway because inhibition of one specific kinase or pathway is not necessarily sufficient to elicit a significant clinical response and may result in rapid resistance. Including the use of a single drug capable of The methods of the present invention can avoid the attack faced by a single drug targeting a single kinase or kinase pathway.

  The method of the present invention is a method of influencing processes important for cell survival, proliferation, growth and malignant transformation, motility and invasion leading to angiogenesis and metastasis by simultaneously targeting multiple protein kinases or protein kinase pathways. including. This method involves the use of a single drug that modulates, modulates or inhibits two or more kinases or kinase pathways.

  Thus, the methods of the invention include methods that include targeting multiple kinases or kinase pathways involved in various diseases, disorders or conditions. In particular, the method contemplated in the present invention involves the use of a single drug that targets the appropriate combination of multiple targets and provides a clinical effect.

  In one embodiment, the method includes administering an effective amount of a single drug that simultaneously targets multiple kinases or kinase pathways to a patient in need of treatment.

  In preferred embodiments, a single drug targets one or more kinases or kinase pathways described in Section 3 below.

  In particularly preferred embodiments, a single drug targets at least one member of the src kinase family of protein kinases. In other preferred embodiments, a single drug targets at least one member of the Rsk kinase family. In still other preferred embodiments, a single drug can target at least one member of the CDK family of protein kinases. In still other preferred embodiments, a single drug targets at least one checkpoint kinase. In other preferred embodiments, the single drug targets at least one member of the MAPK kinase family of protein kinases. The present invention also contemplates the use of a single drug that can target the kinase, which includes ROCK-II, PRK2, PRAK, p70S6 kinase or Aurora A kinase, It is not limited.

  In each of the above embodiments, the present invention contemplates that a single drug simultaneously targets more than one kinase or kinase pathway, preferably more than two kinases or kinase pathways.

  In one embodiment, a single drug targets two or more kinases simultaneously by directly modulating, modulating or inhibiting the activity, expression or function of each specific kinase. In particular, a single drug can simultaneously target multiple kinases or kinase pathways and directly and selectively target specific kinases and / or kinase pathways without affecting other kinases and / or kinase pathways .

  In one embodiment, a single drug can simultaneously modulate, modulate or inhibit multiple kinases or kinase pathways. In certain embodiments, a single drug modulates the activity of multiple kinases, or modulates or inhibits the activity of multiple kinases. In another specific embodiment, a single drug modulates the activity of multiple kinases or modulates or suppresses expression of genes encoding multiple kinases. In yet another specific embodiment, a single drug modulates, modulates or inhibits the function of multiple kinases. In alternative embodiments, a single drug modulates the activity of multiple kinases or modulates or inhibits the activity, expression and / or function of multiple kinases.

  In one embodiment, a single drug modulates the activity of two or more kinases in the same kinase pathway, or modulates or inhibits said kinase. In an alternative embodiment, a single drug modulates, modulates or inhibits multiple kinases in at least two different kinase pathways.

  In another embodiment, a single drug modulates, modulates or inhibits at least two kinases in the same kinase family. In alternative embodiments, a single drug modulates, modulates or inhibits multiple kinases in different kinase families.

  In yet another embodiment, a single drug modulates, modulates or inhibits the corresponding kinase, its downstream target and / or its upstream target.

  In certain embodiments, a single drug modulates, regulates or suppresses multiple genes encoding kinases that are abnormally expressed, abnormally activated or mutated. In this embodiment, the kinase may be overexpressed or underexpressed, or may be overactivated and / or underactivated and / or not activated at all.

In certain embodiments, a single drug targets a group of kinases comprising multiple kinase family members. In one embodiment, a single drug targets a cyclin nucleotide-regulated-phospholipid-regulated kinase and a kinase that is a ribosomal S6 kinase. Examples of kinases that are members of the group are described herein, including protein kinase A (PKA), protein kinase G (PKG) and protein kinase C (PKC), It is not limited to. In another embodiment, the single drug targets Ca ²⁺ / calmodulin kinase. In yet another embodiment of the invention, the single drug targets cyclin dependent kinases. Examples of cyclin dependent kinases are described herein, including cyclin dependent kinase (CDK1), mitogen activated protein kinase (MAPK / ERK) and glycogen synthase kinase (GSK3). However, it is not limited to these. In yet another embodiment of the invention, the single drug targets a protein tyrosine kinase. Examples of tyrosine kinases are described herein, including but not limited to SRC and EFR.

  Histidine kinase can be targeted using the methods of the invention. The kinase includes pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), pyruvate dehydrogenase kinase isoenzyme 3 (PDK3), branched chain α-keto acid dehydrogenase kinase (BCKDK) and pyruvate dehydrogenase kinase isoenzyme (PDK1). However, it is not limited to these.

  In other specific embodiments, the single drug targets serine / threonine kinases. In another embodiment, a single drug targets a kinase that phosphorylates serine / threonine residues near arginine or lysine. In yet another embodiment, the single drug targets a kinase that phosphorylates serine / threonine residues in the proline-rich domain. In another embodiment, the single drug targets a receptor tyrosine kinase. In yet another embodiment, the single drug targets a non-receptor tyrosine kinase. In certain embodiments, a single drug targets DNA-dependent protein kinase (DNA-PK). In yet another embodiment, the single drug targets a kinase that has not been identified in one of the groups, families or subfamilies described herein.

  In a more specific embodiment of the invention, a single drug targets a kinase related to the SRC family of kinases, preferably cSRC, YES, FYN and LCK.

  In another more specific embodiment of the invention, the single drug targets a kinase related to a tyrosine kinase other than an SRC-related kinase. Preferably, the kinase is FES, LYN and SYK.

  In another more specific embodiment, the single drug targets a kinase related to the Rsk family of kinases, preferably targeting Rsk1, Rsk2 and Rsk3.

  In another more specific embodiment, the single drug is a kinase related to the CDK family of kinases, preferably CDK1 / cyclin B1, CDK2 / cyclin A, CDK3 / cyclin E, CDK5 / p53, CDK6 / cyclin D3 and CDK7. Target / cyclin H / MAT1.

  In another more specific embodiment, the single drug targets a kinase associated with the checkpoint family of kinases, preferably CHK1 and CHK2.

  In another more specific embodiment, the single drug targets a kinase associated with the MAPK family of kinases, preferably JNK1, MAPK1 / ERK1, MAPK2 / ERK2, MAPKAP-K5 and MEK1.

  In another more specific embodiment, the single drug targets other kinases identified in Section 3 herein. This kinase includes but is not limited to ROCK-II, PRK2, PRAK, p70S6 kinase and Aurora A.

  In certain embodiments, a single drug targets a kinase or kinase pathway that interacts with each other. In an alternative embodiment, a single drug targets a kinase or kinase pathway that does not interact with each other. In certain other embodiments, a single drug targets a kinase or kinase pathway involved in the same cellular process. In alternative embodiments, a single drug targets a kinase or kinase pathway that is involved in different cellular processes.

  In preferred embodiments, a single drug targets the tyrosine kinase or tyrosine kinase pathway and is useful in the treatment of diseases such as diabetes, cancer, cell proliferative disorders, inflammation and obesity. See also Section 2.

  In a preferred embodiment, the single drug targets at least human CDK1, CDK2, cSRC, Yes, MEK1 and Rsk1. In another preferred embodiment, the single drug has at least 75 activities of CDK1, CDK2, cSRC, Yes, MEK1 and Rsk1 compared to the activity of the kinase under comparable conditions in the absence of a single drug. % Inhibition. In another preferred embodiment, the single drug inhibits the activity of each of CDK1, CDK2, cSRC, Yes, MEK1 and Rsk1 by at least 90% compared to the activity of the kinase under equivalent conditions in the absence of a single drug To do.

  In another embodiment, the single drug exhibits antiproliferative activity in one or more drug resistant cell lines in vitro. This anti-proliferative activity is evidenced by a detectable decrease or decrease in the growth rate of certain proliferative cell lines. In another embodiment, a single drug exhibits anti-cell proliferative activity against a panel of one or more cancer cell lines in vitro. In yet another embodiment, a single drug inhibits various kinases in the in vitro kinase assay described in the examples below.

  In another preferred embodiment, the single drug targets cyclin dependent kinases or cyclin dependent kinase pathways and is useful in the treatment of diseases such as cancer, hyperproliferative diseases and immune diseases.

  In another preferred embodiment, a single drug targets the tyrosine kinase or tyrosine kinase pathway and is useful in the treatment of diseases associated with high or abnormal angiogenesis and angiogenesis. For example, a single drug is useful in the treatment of a cancer or tumor whose growth is promoted by significant angiogenesis or angiogenesis in and around the cancer or tumor.

  In another embodiment, the single drug is cSRC, Yes, Fyn, Lck, Fes, Lyn, Syk, Rsk, CDK1, CDK2, CDK3, CDK5, CDK6, CDK7, CHK1, CHK2, JNK1, MAPK1, MAPK2, MAPKAP- Treatment of diseases related to and targeting at least 2, at least 3, at least 4, at least 5 or at least 7 of the kinase or kinase pathway of K5, MEKI, ROCKII, PRK2, PRAK, p70S6 and Aurora A Useful for. Examples of such diseases include, but are not limited to, cancer or cell proliferation; inappropriate or disease-related angiogenesis; cardiovascular disease; inflammation; insulin resistance, diabetes or obesity; neurological disease; Not.

  In another embodiment, the single drug is Yes, BMX, Syk, Eph, FGFR, RYK, MUSK, JAK1 and EGFR kinase or kinase pathway at least 2, at least 3, at least 4, at least 5, or At least 7 are targeted and useful for the treatment of related diseases. Examples of such diseases include insulin resistance, diabetes or obesity; cancer, cell proliferation and associated congenital syndromes; inflammation; angiogenesis associated with inadequate or disease; cardiovascular disease; or microbial infection, It is not limited to these.

  In another embodiment, the single drug targets at least 2, at least 3, at least 4, or at least 5 of the kinases or kinase pathways of CDK, JNK, ERK, CDKL, ICK, CLK and DYRK. Useful for the treatment of diseases related to Examples of such diseases include, but are not limited to, cancer, cell proliferation and related congenital syndromes; insulin resistance, diabetes or obesity; neurological diseases; or microbial infections.

  In another embodiment, the single drug is at least 2, at least 3, at least 4, at least 5 of the kinase or kinase pathway of Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, DYRK and Yrk. Or at least 7 are targeted and useful for the treatment of diseases associated therewith. Examples of such diseases include, but are not limited to, cancer or hyperproliferation; immunity; inappropriate or disease-related angiogenesis; neurological disease; cardiovascular disease; inflammation; or microbial infection.

  In another embodiment, the single drug is MAPK, MAPK3, ERK2, MAPK7, JNK1, MAPK10, JNK3 alpha or MAPK14 kinase or kinase pathway at least 2, at least 3, at least 4, at least 5, or at least It targets 7 and is useful for the treatment of related diseases. Examples of such diseases include insulin resistance, diabetes or obesity; inflammation; cardiovascular disease; inappropriate or disease-related angiogenesis; cancer, cell proliferation or related congenital diseases; or microbial infections, It is not limited to these.

  In another embodiment, the single drug is CHK1, CHK2, RSK1, RSK2, RSK3, Aurora A, Aurora B, ROCKI, ROCKII or p70S6K kinase or kinase pathway at least 2, at least 3, at least 4, At least 5 or at least 7 are targeted and useful for the treatment of related diseases. Examples of such diseases include insulin resistance, diabetes or obesity; inflammation; inappropriate or disease-related angiogenesis; cardiovascular disease; cancer, hyperproliferation or related congenital diseases; or microbial infections, It is not limited to these.

  In another embodiment, the single drug is pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), pyruvate dehydrogenase kinase isoenzyme 3 (PDK3), branched chain α-keto acid dehydrogenase kinase (BCKDK) or pyruvate dehydrogenase kinase. It targets at least 2 or at least 3 of isoenzyme 1 (PDK1) and is useful in the treatment, prevention, management or amelioration of the diseases associated therewith. Examples of such diseases include, but are not limited to, cancer, hyperproliferation or related congenital diseases; inflammation; angiogenesis; microbial infection; cardiovascular disease; or insulin resistance, diabetes or obesity.

  In another embodiment, a single drug targets ephrin type receptor kinase and neurotrophic type receptor kinase and is useful for the treatment of diseases related thereto. Examples of such diseases include, but are not limited to, neurological disorders. In yet another preferred embodiment, a single drug targets non-receptor tyrosine kinases and IL-1 receptor related kinases and is useful for the treatment of diseases related thereto. Such diseases include, but are not limited to, hematopoiesis, immunity or angiogenesis. In yet another embodiment, the single drug targets at least 2, 3, or at least 4 of the tyrosine kinase, phosphatidylinositol 3-kinase, JNK, IKK and PKC pathways and is associated with diseases associated therewith. Useful for treatment. Examples of said diseases include but are not limited to insulin resistance, diabetes or obesity. In yet another embodiment, the single drug is ABL, EGFR, VEGFR, NGFR, PKC, PDGFR, CDK, MKK1, CHK1 and mTOR kinases or kinase pathways of at least 2, at least 3, at least 4, at least Targeting 5 or at least 7 is useful for the treatment of related diseases. Examples of such diseases include, but are not limited to, cancer or cell proliferation. In yet another embodiment, the single drug targets at least two, at least three or at least four of the kinases or kinase pathways of PKC, Akt, PI-3 kinase, GSK3 and RTK and is associated with diseases related thereto. Useful for treatment. Examples of said diseases include but are not limited to insulin resistance, diabetes or obesity.

  The methods of the invention involve the use of a single drug that is a compound. This single drug includes, but is not limited to, a single drug that targets more than one kinase or kinase pathway. In preferred embodiments, the single drug may be a compound identified as CC001, CC002, CC004 or CC0005.

  The present invention also includes the use of a single drug, also referred to herein as a mixed kinase drug (monotherapy), or one or more drugs that specifically target a single kinase or kinase pathway and / or Intended for use with other treatments (eg, radiation therapy). See Section 4.3 below for a more comprehensive list of other (adjuvant) therapies that can be used in combination with one or more single drugs.

  According to the methods of the present invention, single drugs capable of simultaneously targeting, ie modulating, modulating or inhibiting multiple kinases or kinase pathways critically involved in various diseases or disorders are known in the art Kinase assays such as those described in Examples 4 to 84 below.

1. Patient Populations The present invention provides methods for treating individuals having a disease or condition associated with more than one kinase. As used herein, an “individual”, “patient”, etc. can be a eukaryote, preferably a mammal, more preferably a human.

  In one embodiment, the methods of the invention are useful in the treatment, management or prevention of a disease or disorder in a patient who is refractory or resistant to a single drug that can target one specific kinase or kinase pathway. is there. The methods of the invention are also believed to be useful in the treatment, management or prevention of a disease or disorder in a patient who has received other treatments, currently receiving or who may receive in the future. For example, the compounds of the present invention can be administered to individuals who have been administered other therapeutic agents or have received other treatments (eg, radiation or surgery).

  The methods of the present invention are not only useful for patients who are not receiving treatment, but are also useful in the treatment of patients who are partially or completely refractory to current standard and experimental treatments. In one embodiment, the present invention has been found to be refractory or non-responsive to treatments other than those described above comprising administering a drug that can specifically target a single kinase or kinase pathway. Methods of treatment and prevention are provided for treating or preventing a disease, condition or disorder that may be refractory or refractory or unresponsive. In an alternative embodiment, the present invention has been found to be refractory or non-responsive to treatment comprising administering a single drug or a single drug that can specifically target a kinase pathway or Provided are treatment and prevention methods for treating or preventing diseases, conditions or disorders that may be refractory or non-responsive.

  In another embodiment, the methods of the present invention are currently used in other therapies, such as anti-cancer treatments (eg, chemotherapy, surgery, radiation therapy, antibody treatment, etc.) and anti-inflammatory treatments (eg, steroidal or non-steroidal anti-steroidal). It is useful in the treatment, management or prevention of a disease or disorder in a patient receiving inflammatory drugs, antibody therapy, β-agonist, cytokine therapy, etc.).

  In yet another embodiment, the methods of the invention may be used to treat a disease or disorder in a patient known to be susceptible to a pathological condition, particularly a cancer, obesity or inflammation-related disease, or a disease listed in Section 2. Useful in treatment, management or prevention.

2. Diseases and Disorders The present invention relates to the use of one or more compounds in an animal, preferably a mammal, to prevent, treat or ameliorate symptoms associated with a disease, disorder or infection associated with the activity or inactivity of one or more protein kinases. Includes treatment comprising administration to an animal, most preferably a human. In preferred embodiments, the invention provides at least 2 protein kinases, at least 3 protein kinases, at least 4 protein kinases, at least 5 protein kinases, at least 10 protein kinases or at least 12 protein kinases. It relates to the prevention, treatment or amelioration of symptoms associated with diseases, disorders or inflammation associated with abnormal activity (eg, abnormal upregulation or downregulation). In some embodiments, the methods of the invention are used in combination with one or more treatments. Such treatment includes, but is not limited to, chemotherapy, radiation treatment, hormone treatment and / or biological treatment / immunotherapy.

  The methods of the invention are useful in the treatment, prevention, management or amelioration of various diseases or disorders related to protein kinase activity. Such diseases or disorders include, but are not limited to, diseases associated with gene expression, cytoskeletal integrity, cell adhesion, cell cycle progression, differentiation or metabolism. The disease is controlled by the complex interaction of protein kinases and phosphatases, and the associated dysfunction of cell signaling is linked to many diseases including cancer and diabetes. Sridhar et al., Pharmaceutical Research, 17 (11): 1345-1353 (2000). Therapies that target protein kinases and thus modulate signal transduction have been the subject of intense research. For example, protein kinase C and tyrosine kinase are involved in certain types of cancer, diabetes and complications associated with diabetes. In addition, protein kinase C isoforms are involved in the cellular changes seen in diabetic vascular complications. Sridhar et al., Pharmaceutical Research, 17 (11): 1345-1353 (2000); Charlotte et al., Mol. Endocrinol. 10: 1273-1281 (1996). As a result of the complexity associated with the protein kinase pathway, there is an overlap between various protein kinases and a wide range of diseases or disorders. The methods of the present invention relate to the treatment, management, prevention or amelioration of diseases associated with protein kinases. Such diseases include, but are not limited to, cancer, inflammatory diseases, diabetes, obesity, angiogenic diseases and cardiovascular diseases.

  In particular, the method of the present invention is useful for the prevention, treatment, management and / or amelioration of various diseases. By way of non-limiting example, examples of types of diseases that can be prevented, treated or managed include, but are not limited to, inflammatory conditions. This inflammatory condition includes diabetes (eg, type II diabetes, type I diabetes, diabetes insipidus, diabetes mellitus, adult-onset diabetes, juvenile diabetes, insulin-dependent diabetes, non-insulin-dependent diabetes, malnutrition-related diabetes, Ketosis-prone diabetes or ketosis-resistant diabetes), kidney disorders (eg glomerulonephritis or acute / chronic renal failure), obesity (eg hereditary obesity, dietary obesity, hormone-related obesity or obesity associated with drug administration), hearing loss (Eg hearing loss due to otitis externa or acute otitis media), fibrosis related diseases (eg pulmonary interstitial fibrosis, renal fibrosis, cystic fibrosis, liver fibrosis, wound healing or burn healing (where burns are 1st, 2nd or 3rd degree burns and / or heat, chemical or electrical burns), arthritis (eg, rheumatoid arthritis, rheumatoid spondylitis, degenerative joints) Or gout), allergy, allergic rhinitis, acute respiratory distress syndrome, asthma, bronchitis, inflammatory bowel disease (eg irritable bowel syndrome, mucinous colitis, ulcerative colitis, Crohn's disease, gastritis, esophagitis, Pancreatitis or peritonitis), or autoimmune disease (eg, scleroderma, systemic lupus erythematosus, myasthenia gravis, graft rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis or multiple sclerosis) However, it is not limited to these.

(2.1) Proliferative diseases The methods of the present invention may be used alone or with other treatments known in the art to manage, treat, prevent, inhibit or reduce the occurrence, development, growth or progression of proliferative diseases. Can be used in combination. In a specific embodiment, the method of the present invention, when administered alone or in combination with another cancer treatment known in the art, causes the occurrence, expression, growth of a proliferative disease or condition as measured by the number of affected cells. Or progression over at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70 as compared to the growth or metastasis of the primary tumor in the absence of the methods of the invention. %, At least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20% or at least 10%.

  In one embodiment, the proliferative disease is cancer. Accordingly, the present invention provides methods for the prevention, treatment or management of cancer, inflammation, diabetes, obesity and other kinase related diseases. In a specific embodiment, the method of the present invention, when administered alone or in combination with another cancer treatment known in the art, causes primary tumor growth or cancer cell metastasis in the absence of the method of the present invention. At least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least relative to the growth or metastasis of the primary tumor at 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20% or at least 10% inhibited or reduced.

  In various embodiments, the cancer that can be treated according to the present invention is a head, neck, eye, mouth, throat, esophagus, breast, bone, lung, colon, rectum, stomach, prostate, breast, ovary, testis or other reproductive organ, It may be cancer of the skin, thyroid, blood, lymph nodes, kidney, liver, pancreas and brain or central nervous system.

  Cancers and related diseases that can be treated or prevented by the methods and compositions of the present invention include, but are not limited to, the following diseases. Leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (eg, myeloblastic, proosteoblastic, myelomonocytic, monocytic, erythroleukemia and myelodysplastic) Syndrome), chronic leukemia (non-limiting examples of this are chronic myelocytic (granulocytic) leukemia), chronic lymphocytic leukemia, hairy cell leukemia); Lymphomas, including but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, including smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma Cellular leukemia, solitary plasmacytoma and extramedullary plasmacytoma include but are not limited to; Waldenstrom macroglobulinemia; monoclonal gamma globulin abnormalities with unclear meaning Benign monoclonal gamma globulin abnormality; heavy chain disease; bone and connective tissue sarcoma, examples of this include osteosarcoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant giant cell tumor, bone fibrosarcoma, spondyloma, periosteal sarcoma, soft tissue sarcoma , Angiosarcoma (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, Examples include glioma, astrocytome, brain stem glioma, ventricular epithelioma, oligodendroma, non-glioma, acoustic neuroma, craniopharynoma, medulloblastoma, meningioma, pineal tumor, pineal Breast cancer, including but not limited to, somatoblastoma, primary cerebral lymphoma; adenocarcinoma, lobular (small cell) cancer, intraductal cancer, medullary breast cancer, mucinous breast cancer, tubular breast cancer, Papillary breast cancer, Paget's disease and Adenocarcinoma, including but not limited to, pheochromocytoma and adrenal cancer; Thyroid cancer, including papillary or follicular thyroid cancer Including, but not limited to, medullary thyroid cancer and undifferentiated thyroid cancer; pancreatic cancer, examples of which include, but are not limited to, insulinoma, gastrinoma, glucagonoma, bipoma, somatostatin secreting tumor and carcinoid or islet cell tumor Pituitary cancer, examples of which include, but are not limited to, Cushing's disease, prolactin-secreting tumors, acromegaly and diabetes insipidus; eye cancers, such as eye melanoma (eg, iris) Melanoma, choroidal melanoma and ciliary melanoma) and retinoblastoma; including but not limited to: vaginal cancer, in this case squamous cell carcinoma , Adenocarcinoma and melanoma, including but not limited to: vulvar cancer, examples of which include but are not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease Cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial cancer and uterine sarcoma; ovary Cancers, including but not limited to ovarian epithelial cancer, borderline tumors, germ cell tumors and stromal tumors; esophageal cancers, including squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma , Mucosal epithelial cancer, adenocarcinoma of the adenocarcinoma, sarcoma, melanoma, plasmacytoma, wart cancer and oat cell (small cell) cancer, including but not limited to; (Polypoid), ulceration, surface diffusion, diffuse diffusion, malignant Include, but are not limited to, papilloma, liposarcoma, fibrosarcoma and carcinosarcoma; colon cancer; rectal cancer; liver cancer, examples of which include, but are not limited to, hepatocellular carcinoma and hepatoblastoma; Gallbladder cancer, including but not limited to adenocarcinoma; Cholangiocarcinoma, including but not limited to papillary, nodular and erosive; lung cancer, including Non-small cell carcinoma, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer include but are not limited to: testicular cancer, examples of which include reproductive tumors, seminoma, undifferentiated cancer, Classical (typical), spermatocytes, nonseminoma, embryonic tumors, teratomas, choriocarcinoma (yolk tumor); prostate cancer, in this case adenocarcinoma, smooth Including, but not limited to, myoma and rhabdomyosarcoma; Stem cancer; oral cancer, including but not limited to squamous cell carcinoma; basal cancer; salivary gland cancer; examples include adenocarcinoma, mucosal epithelial cancer and adenoid cystic cancer Pharyngeal cancer, including but not limited to squamous cell carcinoma and wart cancer; skin cancer, including basal cell tumor, squamous cell carcinoma and melanoma, superficial diffuseness Include, but are not limited to, melanoma, nodular melanoma, malignant melanoma, terminal mole melanoma; kidney cancer, examples of this include renal cell carcinoma, adenocarcinoma, adrenal carcinoma, fibrosarcoma, transition Including, but not limited to, epithelial cancer (renal fistula and / or uterus); Wilms tumor; bladder cancer, examples of which include transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma It is not limited. In addition, the cancers include myxosarcoma, osteogenic sarcoma, endothelial sarcoma, lymphatic endothelial sarcoma, mesothelioma, synovial sarcoma, hemangioblastoma, epithelial malignant tumor, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, Sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, malignant sporadic melanoma, sporadic pancreatic cancer, Poissi-Jeggers syndrome, bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, Gastric cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); lymphoid hematopoietic tumors (leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, velvet) Lymphomas), hematopoietic tumors of the myeloid lineage (including acute and chronic myeloid leukemia, including preosteoblastic leukemia), tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma), other tumors ( Including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma ), Central and peripheral nervous system tumors (including astrocytoma, neuroblastoma, glioma and schwanoma), tumors of mesenchymal origin (including fibrosarcoma, rhabdomyosarcoma and osteosarcoma) and other tumors ( Melanoma, xeroderma pigmentosum, keratinous squamous cell tumor, seminoma, follicular thyroid cancer, and teratocarcinoma). Cancers resulting from abnormal apoptosis are also considered to be treated by the methods and compositions of the present invention. Such cancers include, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate and ovary, and precancerous lesions (eg, familial adenomatous polyposis and myelodysplastic syndromes). Not. In a specific embodiment, malignant or abnormal growth changes (eg, metaplasia and bone formation) or hyperproliferative disease in the ovary, bladder, breast, colon, lung, skin, pancreas or uterus are caused by the methods and compositions of the invention. Treated or prevented. In other specific embodiments, sarcoma, melanoma or leukemia is treated or prevented by the methods and compositions of the invention. (For a review of these diseases, see Fishman et al., Medicine, 2nd edition, J. B. Lippincott Co., Philadelphia; and Murphy et al., Informed Decisions: The Complete Book of Cancer, Cancer Dict. (See US Viking Penguin, Penguin Books USA, Inc. (1997)).

(2.2) Inflammatory Diseases, Diabetes and Obesity As mentioned above, abnormal kinase activity is related to inflammatory diseases and diabetes and related effects. Sridhar et al., Pharmaceutical Research, 17 (11): 1345-1353 (2000). Indeed, many kinases are involved in the cellular changes seen in diabetic vascular complications. Charlotte et al., Mol. Endocrinol. 10: 1273-1281 (1996). Furthermore, kinases that interfere with insulin action have been implicated in obesity-related diseases and diabetes because obesity is closely related to the insulin resistance found in type II diabetes. Hirosumi et al., Nature, 420: 333-336 (2002). Obesity and diabetes are also closely related to kinase-mediated chronic inflammatory responses in various signal cascades. P. See 333. Thus, the methods of the present invention relate to the management, treatment or prevention of inflammatory diseases, diabetes, obesity and related diseases. Obesity treated by the methods of the present invention includes hereditary obesity, dietary obesity, obesity associated with drug administration or treatment processes, obesity associated with diabetes. Examples of inflammatory and diabetes related diseases that can be managed, treated, prevented or ameliorated using the methods of the present invention include type I diabetes, juvenile diabetes, type II diabetes (NIDDM), non-insulin dependent diabetes, onset in adulthood Diabetes, myotonic dystrophy, malnutrition related diabetes, ketosis-prone diabetes, keto-resistant diabetes, myotonic dystrophy I, Steiner's disease, hepatic glycogenosis, x-linked type I, liver phosphorylase kinase deficiency, liver phosphorylase kinase deficiency , Glycogenosis VIIIA, x-linked hepatic glycogenosis, phosphorylase kinase, hepatic glycogenosis x-linked type II, glycogenosis IX, glycogenosis VIII, lipoprotein lipase deficiency, lpl deficiency, familial hyperchylomicronemia , Berger-Gruz type hyperlipidemia, essential familial hyperlipidemia, lipase D deficiency, type IA hyperlipoproteinemia, Family micronemia, type I GM2 gangliosidosis, B mutant GM2 gangliosidosis, hexosaminidase A deficiency, Ti-Sachs disease, pseudo-AB mutant Ti-Sachs disease, pancreatic fibrosis, cystic fibrosis Galactose-1-phosphate uridyltransferase deficiency, gal deficiency, classical galactosemia, chronic granulomatosis, type I autosomal cytochrome-b-positive granulomatosis, neutrophil cytoplasmic factor 1 deficiency, soluble oxidase component II Deficiency, soc2 deficiency, p47-phox deficiency, type I Gaucher disease, non-brain juvenile Gaucher disease, glucocerebrosidase deficiency, acid β-glucosidase deficiency, hereditary ironblastic anemia, hereditary iron-enriched anemia, chronic granulation Tumor, X-linked cytochrome-b-negative granulomatous disease, asthma, encephalitis, inflammatory bowel disease Chronic inflammation caused by morbidity, chronic obstructive pulmonary disease (COPD), allergic disease, septic shock, pulmonary fibrosis, non-differentiated spondyloarthropathy, non-differentiated arthropathy, arthritis, inflammatory osteolysis, and chronic viral or bacterial infections Is included, but is not limited thereto.

  Some autoimmune diseases are associated with inflammatory conditions. Thus, what is considered an autoimmune disease and an inflammatory disease overlap. Thus, some autoimmune diseases can also be characterized as inflammatory diseases. Examples of autoimmune diseases that can be managed, treated or prevented using the methods of the present invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison disease, autoimmune diseases of the adrenal gland, autoimmunity Hemolytic anemia, autoimmune hepatitis, autoimmune ovitis and testicularitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, peritoneal sprue dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS) , Chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scar pemphigus, CREST syndrome, cold aggregation disease, Crohn's disease, discoid acne, essential complex cryoglobulinemia, fibromyalgia-fibromyositis , Glomerulonephritis, Craves disease, Guillain-Barre, Hashimoto's disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, obesity Lichen, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type I or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, multiple chondritis Glandular syndrome, rheumatic polymyositis, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma , Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, aortitis syndrome, temporal arteritis, giant cell arteritis, ulcerative colitis, uveitis, vasculitis (eg, herpes dermatitis, pulse Vasculitis), vitiligo, and Wegner granulomatosis, but are not limited to these.

(2.3) Other Diseases—Angiogenesis, Cardiovascular Diseases and Neurological Diseases The present invention relates to the management, treatment, prevention or amelioration of diseases related to kinase activity. For example, a disease or disorder associated with kinase activity can be treated, managed, prevented or ameliorated using the methods of the invention. In one embodiment, the methods of the invention are used to treat angiogenesis-related conditions related to kinase activity. In another embodiment, the methods of the invention are useful for managing, treating, preventing or ameliorating a disease or disorder associated with angiogenesis. In other embodiments, angiogenesis is preferably important for numerous processes such as growth, tissue repair, cancer, psoriasis, diabetic retinopathy and chronic inflammatory diseases in the lungs and joints.

  Thus, in another embodiment, the methods of the invention are used to manage, treat, prevent or ameliorate cardiovascular disease. Such cardiovascular diseases include, but are not limited to, atherosclerosis, restenosis, left ventricular hypertrophy, myocardial infarction, chronic obstructive pulmonary disease or stroke. In yet another embodiment of the invention, the method of the invention is used to manage, treat, prevent or ameliorate a disease or disorder associated with ischemia. Such diseases or disorders include, but are not limited to, ischemic conditions in the heart, kidney, liver or brain as well as ischemia-reperfusion injury resulting from transplantation, surgical trauma, hypertension, thrombus formation or trauma injury. In yet another embodiment, the method of the invention is used to treat a neurodegenerative disease. Examples of said neurodegenerative diseases include, but are not limited to epilepsy, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, peripheral neuropathy, spinal cord injury, AIDS dementia syndrome or Parkinson's disease.

  The methods of the invention are also used to manage, treat, prevent or ameliorate liver disease. Said diseases include but are not limited to hepatitis, alcoholic liver disease, toxic liver disease, steatosis or sclerosis.

(2.4) Diseases Involving Microorganisms Including Bacteria, Viruses and Fungi The methods of the present invention can be used to target kinases involved in infection by microorganisms. This method can be used to prevent host infection by microorganisms. The microorganism includes but is not limited to bacteria, viruses or fungi. The methods of the invention can also be used to treat, prevent or ameliorate symptoms or conditions associated with microbial infections. The microorganism includes but is not limited to bacteria, viruses or fungi. Microorganisms are known in the art, including viruses that can infect organisms and rely on kinases for transmission, survival or homeostasis. Such infectious agents that can be treated, prevented, managed or ameliorated using the methods of the present invention include bacteria (eg, gram positive bacteria, gram negative bacteria, aerobic bacteria, spirochetes, mycobacteria, rickettsia, chlamydia, etc.), parasites Examples include, but are not limited to, insects, fungi (eg, Candida albicans, Aspergillus, etc.), viruses (eg, DNA viruses, RNA viruses, etc.) or tumors. Viral infections that can be treated, prevented, managed or ameliorated include human immunodeficiency virus (HIV); hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus or other hepatitis viruses; cytomegalovirus, Herpes simplex virus-1 (-2, -3, -4, -5, -6), human papillomavirus; respiratory syncytial virus (RSV), parainfluenza virus (PIV), Epstein-Barr virus, metapneumovirus ( MPV) or other viral infections, including but not limited to.

3. Protein Kinases and Kinase Pathways The present invention includes targeting protein kinases. Such protein kinases include, but are not limited to, kinases known in the art and additional kinases that may be discovered. The protein kinases include cyclin nucleotide-regulated phospholipid-regulated kinase and ribosomal S6 kinase (also referred to herein as group “AGC” kinase), Ca ²⁺ / calmodulin kinase (referred to herein as group “CaMK”). ), Cyclin-dependent kinases (referred to herein as group “CMGC” kinases) and protein tyrosine kinases (referred to herein as group “PTK” kinases). Is included, but is not limited thereto. These groups are only given as examples of kinase groups and are not meant to limit the possible groups embodied in the methods of the invention. Indeed, the present invention also relates to methods for targeting kinases outside the four groups described above.

  Kinases that can be targeted using the methods of the invention can also be classified using their functional activity characteristics. For example, the methods of the invention include targeting protein kinases that phosphorylate serine / threonine residues near arginine or lysine residues on a substrate molecule, such as members of the AGC group or CaMK group. In another embodiment, the methods of the invention involve targeting a protein kinase, such as a member of the CMGC group, that phosphorylates serine / threonine residues in the proline-rich domain on the substrate molecule. In yet another embodiment, the methods of the invention include targeting protein receptor kinases that phosphorylate tyrosine residues, such as members of the PTK group. In yet another embodiment, the methods of the invention involve targeting a protein non-receptor kinase, such as a member of the PTK group, that phosphorylates tyrosine residues. Other kinases that can be targeted using the methods of the invention include bispecific kinases, such as kinases that can phosphorylate serine / threonine and tyrosine residues.

  In one embodiment, kinases targeted by the methods of the present invention include, but are not limited to, tyrosine kinases, both receptor tyrosine kinases and non-receptor kilocin kinases. In preferred embodiments, the tyrosine kinase is C-SRC, YES, FYN or LCK. In yet another preferred embodiment, the tyrosine kinase is FES, LYN or SYK. Examples are not intended to limit the possible kinases that can be targeted using the methods of the invention, but other tyrosine kinases include tyrosine-protein kinase (SYK), tyrosine-protein kinase (ZAP-70). Protein tyrosine kinase 2 beta (PYK2), focal adhesion kinase (FAK), B lymphocyte kinase (BLK), hematopoietic cell kinase (HCK), v-yes-1 Yamaguchi sarcoma virus-related oncogene homolog (LYN), T cell specific protein tyrosine kinase (LCK), primary oncogene tyrosine-protein kinase (YES), primary oncogene tyrosine-protein kinase (SRC), primary oncogene tyrosine-protein kinase (FYN), primary oncogene tyrosine-protein Kinase (FGR), Oncogene tyrosine-protein kinase (FER), primary oncogene tyrosine-protein kinase (FES), C-SRC kinase, protein-tyrosine kinase (CYL), tyrosine protein kinase (CSK), megakaryocyte-related tyrosine-protein kinase (CTK) ), Tyrosine-protein kinase receptor (EPH), ephrin type A receptor 1, ephrin type A receptor 4 (EPHA4), ephrin type B receptor (EPHB3), ephrin type A receptor 8 (EPHA8), neurotropic nerve Sex tyrosine kinase receptor type 1 (NTPK1), protein-tyrosine kinase (PTK2), syk-related tyrosine kinase (SRK), protein tyrosine kinase (CTK), tyro3 protein tyrosine kinase (TYRO3), Luton-type agammaglobulinemia tyrosine kinase (BTK), leukocyte tyrosine kinase (LTK), protein-tyrosine kinase (SYK), protein-tyrosine kinase (STY), tek tyrosine kinase (TEK), elk-related tyrosine kinase (ERK) Tyrosine kinase (TIE) having immunoglobulin and egf factor homology domain, protein tyrosine kinase (TKF), neurotropic tyrosine kinase receptor type 3 (NTRK3), mixed series protein kinase 3 (MLK3), protein kinase, mitogenic activity 4 (PRKM4), protein kinase, mitogen activation (PRKM1), protein tyrosine kinase (PTK7), protein tyrosine kinase (EEK), mini brain (Shouji) Drosophila) homolog (MNBH), bone marrow kinase, x-linked (BMX), eph-like tyrosine kinase (ETK1), macrophage stimulating 1 receptor (MST1R), btk-related protein, 135 kd, lymphocyte-specific protein tyrosine kinase (LCK), Fibroblast growth factor receptor 2 (FGFR2), protein tyrosine kinase 3 (TYK3), protein tyrosine kinase (TXK), tec protein tyrosine kinase (TEC), protein tyrosine kinase 2 (TYK2), eph-related receptor tyrosine kinase ligand 1 (EPLG1), t cell tyrosine kinase (EMT), eph tyrosine kinase 1 (EPHT1), zona pellucida receptor tyrosine kinase, 95 kd (ZPK), protein kinase, mitogen activation, kinase 1 (PRKMK1), epe tyrosine kinase 3 (EPHT3), growth arrest specific gene 6 (GAS6), kinase insert domain receptor (KDR), axl receptor tyrosine kinase (AXL), fibroblast growth factor receptor 1 ( FGFR1), v-erb-b2 avian erythroblastic leukemia virus oncogene homolog 2 (FEBB2), fms-like tyrosine kinase 3 (FLT3), neuroepithelial tyrosine kinase (NEP), neurotrophic tyrosine kinase receptor-related 3 ( NTPKR3), epe-related receptor tyrosine kinase ligand 5 (EPLG5), neurotropic tyrosine kinase, receptor, type 2 (NTRK2), receptor-like tyrosine kinase (RYK), tyrosine kinase, b lymphocyte specific (BLK) , Epe tyrosine kinase 2 (EPHT2), ph-related receptor tyrosine kinase ligand 2 (EPLG2), glycogen storage disease VIII, epe-related receptor tyrosine kinase ligand 7 (EPLG7), Janus kinase 1 (JAK1), fms-related tyrosine kinase 1 (FLT1), protein kinase, camp dependent Sex, regulation, type I, alpha (PRKAR1A), wee-1 tyrosine kinase (WEE1), epe-like tyrosine kinase 2 (ETK2), receptor tyrosine kinase musk, insulin receptor (INSR), Janus kinase 3 (JAK3), fms-related tyrosine kinase 3 ligand protein kinase c, beta 1 (PRKCB1), tyrosine kinase type cell surface receptor (HER3), Janus kinase 2 (JAK2), lim domain kinase 1 (LIMK1), bispecific phosphine Atase 1 (DUSP1), hematopoietic cell kinase (HCK), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, eta polypeptide (YWHAH), ret primary oncogene (RET), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, zeta polypeptide (YWHAZ), tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein, beta polypeptide (YWHAB), hepatoma transmembrane kinase (HTK), map kinase kinase 6, Phosphatidylinositol 3-kinase, catalyst, alpha polypeptide (PIK3CA), cyclin-dependent kinase inhibitor 3 (CDKN3), diacylglycerol kinase, del , 130 kd, protein-tyrosine phosphatase, non-receptor type, 13 (PTPN13), abelson murine leukemia virus oncogene homolog 1 (ABL1), diacylglycerol kinase, alpha (DAGK1), focal adhesion kinase 2, epithelial discoidin domain receptor Body 1 (EDDR1), anaplastic lymphoma kinase (ALK), phosphatidylinositol 3-kinase, catalyst, gamma polypeptide (PIK3CG), phosphatidylinositol 3-kinase regulatory subunit (PIK3R1), eph homologous kinase 1 (EHK1), v -Kit hardy-zuckerman4 feline sarcoma virus oncogene homolog (KIT), fibroblast growth factor receptor 3 (FGFR3), vascular endothelial growth factor c (V EGFC), epidermal growth factor receptor 3 (FGFR), oncogene (TRK), growth factor receptor binding protein 7 (GRB7), ras p21 protein activator (RASA2), met primary oncogene (MET), src-like adapter ( SLA), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), nerve growth factor receptor (NGFR), platelet derived growth factor receptor (PDGFR), platelet derived growth factor receptor beta (PDGFRB) Bispecific tyrosine- (Y) -phosphorylated regulatory kinase 2 (DYRK2), bispecific tyrosine- (Y) -phosphorylated regulatory kinase 3 (DYRK3), bispecific tyrosine- (Y) -phosphorus Oxidation-regulated kinase 4 (DYRK4), bispecific tyrosine- (Y) -phosphorylation-regulated kinase 1A (D RK1A), bispecific tyrosine- (Y) -phosphorylation regulated kinase 1B (DYRK1B), CDC-like kinase 1 (CLK1), protein tyrosine kinase STY, CDC-like kinase 4 (CLK4), CDC-like kinase 2 (CLK2) And CDC-like kinase 3 (CLK3).

  In another embodiment, the methods of the invention are used to target serine / threonine kinases. By way of example and without intending to limit the possible kinases that can be targeted using the methods of the present invention, serine / threonine kinases or related molecules include cyclin dependent kinases (CDK7), rac serine / threonine proteins. Kinase, serine-threonine protein kinase n (PKN), serine / threonine protein kinase 2 (STK2), zipper protein kinase (ZPK), protein-tyrosine kinase (STY), Bruton's agammaglobulinemia tyrosine kinase (BTK) , Mkn28 kinase, protein kinase, x-linked (PRKX), elk-related tyrosine kinase (ERK), ribosomal protein s6 kinase, 90 kd, polypeptide 3 (RPS6KA3), glycogenosis VIII, death-related protein kina Ze I (DAPK1), pctaire protein kinase 1 (PCTK1), protein kinase, interferon-induced double-stranded rna (PRKR), activin a receptor, type II-like kinase 1 (ACVRLK1), protein kinase, camp-dependent, catalytic , Alpha (PRKACA), protein kinase, y-linked (PRKY), G protein-coupled receptor kinase 2 (GPRK21), protein kinase c, theta form (PRKCQ), lim domain kinase 1 (LINK1), phosphoglycerate kinase 1 ( PGK1), lim domain kinase 2 (LIMK2), c-jun kinase, activin a receptor, type II-like kinase 2 (ACVRLK2), Janus kinase 1 (JAK1), elkl motif kinase (EMK1), male cell associated kinase (MAK), casein kinase 2, alpha-prime subunit (CSNK2A2), casein kinase 2, beta polypeptide (CSNK2B), casein kinase 2, alpha 1 polypeptide (CSNK2A1), ret primary Oncogene (RET), hematopoietic ancestral kinase 1, conserved helix loop helix ubiquitous kinase (CHUK), casein kinase 1, delta (CSNK1D), casein kinase 1, epsilon (CSNK1E), v-akt murine thymoma Viral oncogene homolog 1 (AKT1), tumor protein p53 (TP53), protein phosphatase 1, regulatory (inhibitor) subunit 2 (PPP1R2), oncogene pim-1 (PIM1), transform Ming growth factor beta receptor, type II (TGFBR2), transforming growth factor beta receptor, type I (TGFBR1), v-raf murine sarcoma virus oncogene homolog b1 (BRAF), bone morphogenetic receptor type II (BMPR2) ), V-raf murine sarcoma 3611 virus oncogene homolog 1 (ARAF1), v-raf murine sarcoma 3611 virus oncogene homolog 2 (ARAF2), protein kinase C (PKC), v-kit hardy-zuckerman 4 feline sarcoma virus oncogene Homolog (KIT) and c-KIT receptor (KITR) are included, but are not limited to these.

  In one embodiment, kinases targeted using the methods of the invention include cyclin dependent or kinases from the CDK family of kinases. Although the present invention embodies the modulation of members of the CDK family of kinases, in certain embodiments the CDK is CDK1 / cyclin B1, CDK2 / cyclin A, CDK3 / cyclin E, CDK5 / p35, CDK6 / cyclin D3 or CDK7 / Cyclin H / MAT1. Examples are not intended to limit the possible kinases that can be targeted using the methods of the invention, but other members of the CDK family of kinases targeted using the methods of the invention are cyclin dependent. Sex kinase 2 (CDK2), cyclin-dependent kinase 7 (CDK7), CDK-activated kinase (CAK), THIIH basic transcription factor complex kinase subunit, 39 kDa protein kinase, STK1, CAK1, cyclin-dependent kinase 6 (CDK6), Cell division control 2 protein (CDC2), p34 protein kinase, cyclin dependent kinase 1 (CDK1), cell division protein kinase 1, cell division protein kinase 2 (CDK2), cyclin dependent protein kinase, p33 protein kinase, cell division tag Protein kinase (CDK3), cyclin-dependent protein kinase 3, cell division protein kinase 5 (CDK5), cyclin-dependent protein kinase 5, tau protein kinase II (TPKII), serine / threonine protein kinase (PSSALRE), serine / threonine Protein kinase PCTAIRE-1 (PCTAIRE1), serine / threonine protein kinase (PCTAIRE-2), serine / threonine protein kinase PFTAIRE-1 (PFTAIRE1), serine / threonine protein kinase ALS2CR7 (PFTAIRE2), cell division protein kinase 4 (CDK4) Cyclin-dependent kinase 4, PSK-J3, cell division protein kinase 6, serine / threonine tongue Protein kinase (PLSTIRE), cell division protein kinase 10 (CDK10), cyclin-dependent protein kinase 10, serine / threonine protein kinase (PISSLRE), serine / threonine kinase CDC2L1 (PITSLRE), galactosyltransferase-related protein kinase p58 / GTA, Cell division cycle 2 like 1 (CLK-1), CDK11, p58CLK-1, Cell division protein kinase 9 (CDK9), Cyclin-dependent protein kinase 9, Serine / threonine protein kinase PITALRE, C2K, Cell division cycle 2 like 5 ( Cholinerase-related cell division controller (CHED), CDC2L, cell division cycle 2-related protein kinase 7 (CRK7), CDC2-related tamper Protein kinase 7, cell division protein kinase 8 (CDK8), protein kinase K35, cyclin-dependent kinase-like 1 (CDC2-related kinase) (CDKL1), CDC2-related kinase, serine / threonine protein kinase, cyclin-dependent kinase-like 2 ( CDC2-related kinase) (CDKL2), CDC2-related kinase, p56 protein kinase, cyclin-dependent kinase-like 3 (CDKL-3), cyclin-dependent kinase-like 5 (CDKL-5), glycogen synthase kinase 3 alpha (GSK3 alpha), Examples include, but are not limited to, glycogen synthase kinase 3 beta (GSK3 beta) and intestinal cell (MAK-like) kinase (ICK). Many of these cyclin-dependent kinases have been shown to be involved in cell signaling pathways involved in various physiological states. Such conditions include, but are not limited to, cancer, hyperproliferative diseases and immune diseases.

  In one embodiment of the invention, the kinase targeted using the methods of the invention includes MAP kinase. Although the present invention embodies the modulation of members of the MAP family of kinases, in preferred embodiments the MAP kinase is JNK1, MAPK1 / ERK1, MAPK2 / ERK2, MAPKAP-K5 or MEK1. Examples are not intended to limit the possible kinases that can be targeted using the methods of the invention, but other members of the MAP kinase family of kinases targeted using the methods of the invention include mitogens. Activated protein kinase 3 (MAPK3), p44erk1, p44mapk, mitogen activated protein kinase (MAP kinase 3; p44), ERK1, PRKM3, P44ERK1, P44MAPK, mitogen activated protein kinase 1 (MAPK1), mitogen activated protein kinase kinase 1 (MEK1), MAP2K protein tyrosine kinase ERK2, mitogen-activated protein kinase 2, extracellular signal-regulated kinase 2, protein tyrosine kinase ERK2, mitogen-activated protein Kinase 2, extracellular signal-regulated kinase 2, ERK, p38, p40, p41, ERK2, ERT1, MAPK2, PRKM1, PRKM2, P42MAPK, p41mapk, mitogen activated protein kinase (MAPK7), BMK1 kinase, extracellular signal-regulated kinase 5 , BMK1, ERK4, ERK5, PRKM7, nemo-like kinase (NLK), promising ortholog of mouse nemo-like kinase, mitogen-activated protein kinase 8 (MAPK8), protein kinase JNK1, JNK1 beta protein kinase, JNK1 alpha protein kinase, c -Jun N-terminal kinase 1, stress-activated protein kinase JNK1, JNK, JNK1, PRKM8, SAPK1, JNK1A2, JNK2 1B1 / 2, mitogen activated protein kinase 10 (MAPJ10), c-Jun kinase 3, JNK3 alpha protein kinase, c-Jun N-terminal kinase 3, stress activated protein kinase JNK3, stress activated protein kinase beta, mitogen activated Protein kinase 9 (MAPK9), MAP kinase 9, c-Jun kinase 2, c-Jun N-terminal kinase 2, stress-activated protein kinase JNK2, JNK2, JNK2A, JNK2B, PRKM9, JNK-55, JNK2BETA, p54aSAPK, JNK2ALPHA, Mitogen-activated protein kinase 14 (MAPK14), p38 MAP kinase, MAP kinase Mxi2, Csaids binding protein, MAX interaction Protein 2, stress activated protein kinase 2A, p38 mitogen activated protein kinase, cytokine inhibitory anti-inflammatory drug binding protein, RK, p38, EXIP, Mxi2, CSBP1, CSBP2, CSPB1, PRKM14, PRKM15, SAPK2A, p38ALPHA, mitogen activated Protein kinase 11 (MAPK11), stress activated protein kinase 2, stress activated protein kinase 2b, mitogen activated protein kinase p38-2, mitogen activated protein kinase p38 beta, P38B, SAPK2, p38-2, PRKM11, SAPK2B, p38Beta, P38BETA2, mitogen-activated protein kinase 13 (MAPK13), stress activation Protein kinase 4, mitogen activated protein kinase p38 delta, SAPK4, PRKM13, p38 delta, mitogen activated protein kinase 12 (MAPK12), p38 gamma, stress activated protein kinase 3, mitogen activated protein kinase 3, ERK3, ERK6 , SAPK3, ERKM12, SAPK-3, P38GAMMA, mitogen activated protein kinase 6 (MAPK6), MAP kinase isoform p97, mitogen activated 5 protein kinase, mitogen activated 6 protein kinase, extracellular signal-regulated kinase 3, extracellular signal Regulatory kinase, p97, ERK3, PRKM6, p97MAPK, mitogen-activated protein kinase 4 (MAPK4), Erk3 Examples include, but are not limited to, ream protein kinases, mitogen activated 4 protein kinases (MAP kinase; p63), PRKM4, p63MAPK, ERK3-RELATED and extracellular signal-regulated kinase 8 (ERK7).

  In one embodiment, kinases targeted using the methods of the invention include Rsk kinase. Although the present invention embodies the modulation of members of the Rsk family of kinases, in a more preferred embodiment the Rsk kinase is Rsk1, Rsk2 or Rsk3. Examples are not intended to limit the possible kinases that can be targeted using the methods of the present invention, but other members of the RSK family of kinases targeted using the methods of the present invention include ribosomal proteins S6 kinase-like 1 (RskL2), ribosomal protein S6 kinase, 52 kDa, polypeptide 1 (RskL2), ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (Rsk3), ribosomal protein S6 kinase, 90 kDa, polypeptide 6 (Rsk4), Ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (Rsk2), ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (Rsk1 / p90Rsk), p70 ribosomal S6 kinase beta, ribosomal protein S6 kinase 70 kDa, polypeptide 2 (p70S6Kβ), serine / threonine kinase 14 alpha, ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (p70S6K), ribosomal protein S6 kinase, 90 kDa, polypeptide 4, ribosomal protein kinase B, mitogen and stress activity Protein kinase 2 (MSK2), mitogen and stress activated protein kinase 1 and ribosomal protein S6 kinase, 90 kDa, polypeptide 5 (MSK1), but are not limited to these.

  In one embodiment of the invention, the targeted kinase includes a kinase from the CHK family. Although the present invention embodies the modulation of CHK family members, in a preferred embodiment the CHK kinase is CHK1 or CHK2. By way of example and not intended to limit the possible kinases that can be targeted using the method of the invention, other members of the checkpoint family targeted using the method of the invention include serine / threonine. Protein kinase (CHK1), serine / threonine kinase 11 (LKB1), PAS-serine / threonine kinase (PASK), serine / threonine protein kinase PIM-2, primary oncogene serine / threonine protein kinase PIM-1 and primary oncogene serine / Including, but not limited to, threonine protein kinase PIM-3.

  Other families of kinases that can be targeted using the methods of the invention include Rho-related coiled-coil containing protein kinases p160ROCK (ROCK1), Rho kinase (ROCK2), mitogen-activated protein kinase activated protein kinase 5 (PRAK), ribosome Examples include, but are not limited to, protein S6 kinase (p70S6α), ribosomal protein S6 kinase beta 2 (p70S6β), serine / threonine protein kinase 15 (Aurora A) and serine / threonine protein kinase 12 (Aurora B).

4). Therapeutic / Prophylactic Protocols and Regimens (4.1) Methods The present invention relates to the use of one or more single drugs in the treatment of a disease, disorder or condition that is at least partially related to the activity of at least two different kinases. Is included.

  In one embodiment, the present invention is at least two of CDK1, CDK2, cSRC, Yes, MEK1, and Rsk1. A method of modulating the activity of a plurality of kinases comprising contacting the plurality of kinases with an amount of a single drug sufficient to detectably change the activity of the kinase is provided. In a preferred embodiment, the present invention relates to the activity of the kinase comprising contacting at least three of the kinases CDK1, CDK2, cSRC, Yes, MEK1 and Rsk with a single drug under equivalent conditions in the absence of the single drug. A method of inhibiting at least 75% compared to the activity of said kinase. In a specific embodiment, the single drug is CC001 or CC004. In another preferred embodiment, the present invention relates to the activity of each of the kinases described above comprising contacting at least three of the kinases CDK1, CDK2, cSRC, Yes, MEK1 and Rsk with a single drug without the presence of a single drug. Methods of inhibiting at least 90% compared to the activity of the kinase under conditions are provided. In a specific embodiment, the single drug is C0001.

  In another embodiment, the single drug exhibits antiproliferative activity in one or more drug resistant cell lines in vitro. This antiproliferative activity is demonstrated by a detectable decrease or decrease in the growth rate of a particular proliferating cell line. In another embodiment, the single drug exhibits antiproliferative activity against a panel of one or more cancer cell lines in vitro. In yet another embodiment, the single drug inhibits various kinases in the in vitro kinase assay in the Examples section below.

  In another preferred embodiment, the single drug targets cyclin dependent kinases or cyclin dependent kinase pathways and is useful in the treatment of diseases such as cancer, hyperproliferative diseases and immune diseases.

  In another preferred embodiment, the single drug targets the tyrosine kinase or tyrosine kinase pathway and is useful in the treatment of diseases involving high or abnormal angiogenesis and angiogenesis. For example, a single drug is useful in the treatment of a cancer or tumor whose growth is promoted due to high angiogenesis and angiogenesis in and around the cancer or tumor.

  In another embodiment, the present invention relates to cSRC, Yes, Fyn, Lck, Fes, Lyn, Syk, Rsk, CDK1, CDK2, CDK3, CDK5, CDK6, CDK7, CHK1, CHK2, JNK1, MAPK1, MAPK2, MAPKAP- Contacting a plurality of kinases comprising two or more of K5, MEK1, ROCKII, PRK2, PRAK, p70S6 or Aurora A with a single drug at a concentration sufficient to detectably modulate the activity of the kinase Methods are provided for modulating the activity of multiple kinases by comparing the activity of the kinase with that of the kinase under comparable conditions in the absence of a single drug. In a specific embodiment, said contact is cancer or cell proliferation in vivo; inappropriate or disease-related angiogenesis; cardiovascular disease; inflammation; insulin resistance, diabetes or obesity; neurological disease; or a microbial infection At a concentration sufficient to treat the condition in an individual suffering from. In another embodiment, the present invention relates to a condition that is cancer or cell proliferation; angiogenesis associated with inadequate or disease; cardiovascular disease; inflammation; insulin resistance, diabetes or obesity; neurological disease; CSRC, Yes, Fyn, Lck, Fes, Lyn, Syk, Rsk, CDK1, CDK2, CDK3, CDK5, CDK6, CDK7, CHK1, CHK2, JNK1, MAPK1, MAPK2, MAPKAP-K5, MEK1 , ROCKII, PRK2, PRAK, p70S6 or Aurora A. A method of treating said individual is provided comprising administering a single drug in an amount sufficient to modulate two or more activities of Aurora A.

  In another embodiment, the present invention provides a method for detectably modulating a plurality of kinases comprising two or more of Yes, BMX, Syk, Eph, FGFR, RYK, MUSK, JAK1 or EGFR. Provided are methods for modulating the activity of multiple kinases as compared to the activity of the kinase under comparable conditions in the absence of a single drug comprising contacting with a sufficient concentration of the single drug. In a specific embodiment, said contact is in vivo insulin resistance, diabetes or obesity; cancer, cell proliferation or related congenital syndromes; inflammation; angiogenesis associated with inappropriate or disease; cardiovascular disease; or by microorganisms Performed at a concentration sufficient to treat the condition in an individual suffering from the condition being infected. In another embodiment, the present invention relates to insulin resistance, diabetes or obesity; cancer, cell proliferation or related congenital syndromes; inflammation; angiogenesis associated with inadequate or disease; cardiovascular disease; or microbial infection Administer an amount of a single drug sufficient to modulate two or more activities of Yes, BMX, Syk, Eph, FGFR, RYK, MUSK, JAK1 and EGFR to an individual suffering from a condition A method for treating said individual.

  In another embodiment, the present invention provides a kinase at a concentration sufficient to detectably modulate a plurality of kinases comprising two or more of CDK, JNK, ERK, CDKL, ICK, CLK and DYRK. Methods are provided for modulating the activity of a plurality of kinases as compared to the activity of the kinase under comparable conditions in the absence of a single drug, including contacting with a single drug. In a specific embodiment, said contacting is said to be in an individual suffering from a condition that is in vivo cancer, cell proliferation or a congenital syndrome associated therewith; insulin resistance, diabetes or obesity; neurological disease; or infection by microorganisms. Perform at a concentration sufficient to treat. In another embodiment, the invention provides a CDK for an individual suffering from a condition that is cancer, cell proliferation or a congenital syndrome associated therewith; insulin resistance, diabetes or obesity; neurological disease; There is provided a method of treating said individual comprising administering an amount of a single drug sufficient to modulate two or more activities of JNK, ERK, CDKL, ICK, CLK and DYRK.

  In another embodiment, the present invention detectably modulates a plurality of kinases comprising two or more of Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, DYRK and Yrk. There is provided a method of modulating the activity of a plurality of kinases as compared to the activity of the kinase under equivalent conditions in the absence of a single drug comprising contacting with a single drug at a concentration sufficient for. In a specific embodiment, said contact is in vivo in an individual suffering from a condition that is cancer or hyperproliferation; immunity; inappropriate or disease-related angiogenesis; neurological disease; cardiovascular disease; inflammation; or microbial infection Performed at a concentration sufficient to treat the condition. In another embodiment, the invention relates to an individual suffering from a condition that is cancer or hyperproliferation; immunity; inappropriate or disease-related angiogenesis; neurological disease; cardiovascular disease; inflammation; or microbial infection. Administering a sufficient amount of a single drug to modulate the activity of two or more of Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, DYRK and Yrk I will provide a.

  In another embodiment, the invention is sufficient to detectably modulate a plurality of kinases comprising two or more of MAPK, MAPK3, ERK2, MAPK7, JNK1, MAPK10, JNK3 alpha or MAPK14. A method is provided for modulating the activity of a plurality of kinases as compared to the activity of the kinase under equivalent conditions in the absence of a single drug comprising contacting with a single concentration of a single drug. In a specific embodiment, the contact is in vivo insulin resistance, diabetes or obesity; inflammation; cardiovascular disease; inappropriate or disease-related angiogenesis; cancer, cell proliferation or related congenital disease; or microbial infection In an individual suffering from a condition that is sufficient to treat the condition. In another embodiment, the invention relates to insulin resistance, diabetes or obesity; inflammation; cardiovascular disease; inappropriate or disease-related angiogenesis; cancer, cell proliferation or related congenital disease; or infection by microorganisms. Administering an amount of a single drug sufficient to modulate the activity of two or more of MAPK, MAPK3, ERK2, MAPK7, JNK1, MAPK10, JNK3 alpha or MAPK14 to an individual suffering from a condition A method of treating said individual comprising.

  In another embodiment, the invention provides a plurality of kinases comprising two or more of CHK1, CHK2, RSK1, RSK2, RSK3, Aurora A, Aurora B, ROCKI, ROCKII, or p70S6K, wherein the activity of the kinase is detected. A method is provided for modulating the activity of a plurality of kinases as compared to the activity of the kinase under equivalent conditions in the absence of a single drug comprising contacting with a single drug at a concentration sufficient to rate. In a specific embodiment, said contacting is in vivo insulin resistance, diabetes or obesity; inflammation; angiogenesis associated with inappropriate or disease; cardiovascular disease; cancer, hyperproliferative disease or related congenital disease; or microorganism In an individual suffering from a condition which is an infection due to, at a concentration sufficient to treat said condition. In another embodiment, the invention relates to insulin resistance, diabetes or obesity; inflammation; angiogenesis associated with inappropriateness or disease; cardiovascular disease; cancer, hyperproliferative disease or related congenital disease; or by microorganisms A single amount of an amount sufficient to modulate two or more activities of CHK1, CHK2, RSK1, RSK2, RSK3, Aurora A, Aurora B, ROCKI, ROCKII or p70S6K in an individual suffering from an infectious condition There is provided a method of treating said individual comprising administering a drug.

  In another embodiment, the present invention provides pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), pyruvate dehydrogenase kinase isoenzyme 3 (PDK3), branched chain α-keto acid dehydrogenase kinase (BCKDK) or pyruvate dehydrogenase kinase isoenzyme. Equivalent conditions in the absence of a single drug comprising contacting a plurality of kinases comprising two or more of enzyme 1 (PDK1) with a single drug at a concentration sufficient to detectably modulate the activity of the kinase A method for modulating the activity of a plurality of kinases relative to the activity of the kinase in is provided. In a specific embodiment, said contact is in vivo suffering from a condition that is cancer, hyperproliferative disease or related congenital disease; inflammation; angiogenesis; microbial infection; cardiovascular disease; or insulin resistance, diabetes or obesity At a concentration sufficient to treat the condition in an individual. In another embodiment, the invention suffers from a condition that is cancer, hyperproliferative disease or related congenital disease; inflammation; angiogenesis; microbial infection; cardiovascular disease; or insulin resistance, diabetes or obesity. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), pyruvate dehydrogenase kinase isoenzyme 3 (PDK3), branched chain α-keto acid dehydrogenase kinase (BCKDK) or pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) A method of treating said individual comprising administering a single drug in an amount sufficient to modulate two or more activities of

  In another embodiment, the present invention is sufficient to modulate two or more activities of CDK1, CDK2, cSrc, Yes, Rsk1 or MEK1 on an individual suffering from a condition that is cancer or a proliferative disease. There is provided a method of treating said individual comprising administering an amount of a single drug.

  In another embodiment, a single drug targets ephrin type receptor kinase and neurotrophic type receptor kinase and is useful, for example, in the treatment of (but not limited to) neurological disorders. In another preferred embodiment, a single drug targets non-receptor tyrosine kinases and IL-1 receptor related kinases and is useful in the treatment of diseases associated with, for example, but not limited to hematopoiesis, immunity or angiogenesis It is. In yet another embodiment, the single drug targets at least 2, at least 3 or at least 4 of the tyrosine kinase, phosphatidylinositol 3 kinase, JNK, IKK and PKC pathways, eg, insulin resistance, diabetes or Useful for treating diseases associated with but not limited to obesity. In yet another embodiment, the single drug is ABL, EGFR, VEGFR, NGFR, PKC, PDGFR, CDK, MKK1, CHK1 and mTOR kinase or kinase pathway at least 2, at least 3, at least 4, Target at least 5 or at least 7 and are useful, for example, to treat diseases associated with, but not limited to, cancer or cell proliferation. In yet another embodiment, the single drug targets at least 2, at least 3, or at least 4 of the kinase or kinase pathway of PKC, Akt, PI-3 kinase, GSK3 and RTK, such as, but not limited to Useful for treating diseases associated with insulin resistance, diabetes or obesity.

  In another embodiment, the present invention provides a method for inhibiting the activity of a plurality of kinases as described above comprising contacting a plurality of kinases with a single drug, wherein at least two activities of the plurality of kinases described above are detectable At least 20%, 25%, 30%, 35%, 40% compared to at least two activities of the multiple kinases under equivalent conditions in which no single drug is present by a single drug, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% inhibited. In a more specific embodiment, the plurality of kinases comprises CDK1, CHK2, PRK2 and ROCK-II and the single drug is CDK1, CHK2, PRK2 and in an in vitro kinase assay in which a single drug is present at a concentration of about 3 μM. ROCK-II inhibits at least 90% compared to at least two activities of the kinase under comparable conditions in the absence of a single drug. In another specific embodiment, the single drug is at least 7 of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. Inhibits 90% or more compared to at least 7 activities of the kinases listed in Table 2. In another specific embodiment, the single drug is at least 12 of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. Inhibits 75% or more compared to at least 12 activities of the kinases listed in Table 2. In another specific embodiment, the single drug is at least 21 of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. Inhibits 50% or more compared to at least 21 activities of the kinases listed in Table 2.

  In another embodiment, the invention provides a method of treating an individual comprising administering to a subject suffering from a condition a therapeutically effective amount of a single drug that detectably inhibits the activity of multiple kinases in vivo. provide. In a specific embodiment, the plurality of kinases are cSRC, Yes, Fyn, Lck, Fes, Lyn, Syk, Rsk, CDK1, CDK2, CDK3, CDK5, CDK6, CDK7, CHK1, CHK2, JNK1, MAPK1, MAPK2, MAPLAP- K5, MEK1, ROCKII, PRK2, PRAK, p70S6 or Aurora A; Yes, BMX, Syk, Eph, FGFR, RYK, MUSK, JAK1 or EGFR; CDK, JNK, ERK, CDKL, ICK, CLK or DYRK; Src, Yes , Fyn, Lyn, Lck, Blk, Hck, Fgr, DYRK and Yrk; MAPK, MAPK3, ERK2, MAPK7, JNK1, MAPK10, JNK3 alpha or MAPK14; CHK1, CHK2, R K1, RSK2, RSK3, Aurora A, Aurora B, ROCK1, ROCKII or p70S6K; pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), pyruvate dehydrogenase kinase isoenzyme 3 (PDK3), branched chain α-keto acid dehydrogenase kinase ( BCKDK) or pyruvate dehydrogenase kinase isoenzyme 1 (PDK1); or CDK1, CDK2, cSrc, Yes, Rsk1 or MEK1. In another specific embodiment, a single drug inhibits at least one activity of a plurality of kinases as described above, ie compared to at least two activities of the kinase under equivalent conditions in the absence of a single drug. At least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% Inhibit. In another specific embodiment, said condition is insulin resistance, diabetes or obesity; inflammation; inappropriate or disease-related angiogenesis; cardiovascular disease; cancer, hyperproliferative disease or related congenital disease; or microbial infection One or more of In a more specific embodiment, the plurality of kinases is CDK1, CHK2, PRK2 and ROCK-II, and the single drug is CDK1, CHK2, PRK2 and in an in vitro kinase assay in which a single drug is present at a concentration of about 3 μM. Inhibits ROCK-II by at least 90% compared to the activity of the CDK1, CHK2, PRK2 and ROCK-II under comparable conditions in the absence of a single drug. In another specific embodiment, the single drug has at least 7 activities of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. More than 90% inhibition compared to at least 7 activities of the kinases listed in Table 2. In another specific embodiment, the single drug has at least 12 activities of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. More than 75% inhibition compared to at least 12 activities of the kinases listed in Table 2. In another specific embodiment, the single drug has at least 21 activities of the kinases listed in Table 2 in an in vitro kinase assay in which the single drug is present at a concentration of about 3 μM under equivalent conditions in the absence of the single drug. Inhibit at least 50% compared to at least 21 activities of the kinases listed in Table 2.

  In one embodiment, the present invention provides a method of treating an individual having one or more pathological conditions comprising administering an effective amount of compound CC001, wherein the effective amount detects the activity of one or more kinases. One or more kinases that are capable of being modulated are associated with one or more pathological conditions. In a specific embodiment, the kinase is CDK1 / cyclin B, CDK2 / cyclin A, cSRC, Yes, MEK or Rsk1. In another embodiment, the invention provides a method of treating an individual having one or more pathological conditions comprising administering an effective amount of compound CC004, said effective amount comprising the activity of one or more kinases. One or more kinases that are detectably modulated and are associated with one or more pathological conditions. In a specific embodiment, the kinase is CDK1 / cyclin B, CDK2 / cyclin A, cSrc, Yes, MEK or Rsk1. In another specific embodiment, said treatment comprises administering both CC001 and CC004 to the individual.

(4.2) Compounds Currently, four compounds designated CC001, CC002, CC004 and CC005 (see Example 1) have been identified as acting as a single drug. These four compounds have been found to modulate, in particular inhibit, the activity of several kinases, including Src family kinases and CDK kinases.

  As described below, any of the single drugs described above can be administered to an individual in combination with one or more treatments associated with the particular pathological condition to be treated. Thus, the present invention also provides a method for treating an individual having one or more pathological conditions comprising administering an effective amount of compound CC004 or CC001 in combination with a second compound that is a compound other than the single drug. However, the effective amount detectably modulates the activity of one or more kinases, wherein the one or more kinases are associated with one or more pathological conditions.

  Of course, each of the two compounds described above can be combined with other single drugs in either a therapeutic regimen that includes only a single drug or a regimen that includes other auxiliary drugs.

  Examples of second auxiliary drugs useful in the treatment of certain pathological conditions are listed below.

The present invention provides for the use of preferred single drugs to target multiple kinases and to treat diseases, disorders and conditions that can be treated by targeting multiple kinases. In one embodiment, preferred single drugs for targeting multiple kinases are indazole compounds having formula (Ia), and isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrations thereof. It is a thing.

In the above formula,
R ¹ is —H, —halo, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , — ^{C (O) R 5, -OC} (O) R 5, -NHC (O) R 5, -SO 2 R 6, - aryl, - heterocycle, - heteroaryl, - cycloalkyl, - _{(C 1-6} Alkylene) —R ² or —O— (C _1-6 alkylene) -R ² ;
R ² represents —H, —halo, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , — ^{C (O) R 5, -OC} (O) R 5, -NHC (O) R 5, -SO 2 R 6, - aryl, - heterocyclic, - cycloalkyl - heteroaryl or;
R ³ is independently -H, -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, -C _1-6 haloalkyl, -cycloalkyl, -aryl or -heterocycle;
Each R ⁴ is independently —H, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —cycloalkyl, —aryl, —heterocycle or — (C _1-6 alkylene); -OR ³ ;
R ⁵ is —H, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —cycloalkyl, —aryl, —heterocycle, —OR ³ or —N (R ⁴ ) ₂ . Yes;
R ⁶ is -H, -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, -N (R ⁴ ) ₂ , -cycloalkyl, -aryl or -heterocycle, -C (R < ⁷ >)-or -N- (wherein up to three Z can be -N-);
R ⁷ is —H, —halo, —C _1-6 alkyl, —C _1-6 haloalkyl, —O— (C _1-6 haloalkyl), —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , —C (O) R ⁵ , —OC (O) R ⁵ , —NHC (O) R ⁵ , —SO ₂ R ⁶ , —aryl, - heterocycle, - cycloalkyl, -C (O) NH- _{(C 1-6 alkylene) n} - cycloalkyl, -C (O) NH- _{(C 1-6 alkylene) n} - aryl, -C (O) NH- (C _1-6 alkylene) _n -heterocycle,-(C _1-6 alkylene) -cycloalkyl,-(C _1-6 alkylene) -aryl,-(C _1-6 alkylene) -heterocycle,- O- (C _1-6 alkylene) -C (O) R ⁵ , —O— (C _1-6 alkylene) ) —N (R ⁴ ) ₂ , —O (C _1-6 alkylene) -cycloalkyl, —O— (C _1-6 alkylene) -aryl or —O— (C _1-6 alkylene) -heterocycle (here R ⁷ is attached to the bicyclic ring system via a carbon atom, and n is 0 or 1);
R ⁹ is —H, —C _1-6 alkyl or cycloalkyl.

In one embodiment, each -Z is -C ^{(R 7)} - is.

In another embodiment, R ¹ is —H.

In yet another embodiment, R ¹ is —C _1-6 alkyl.

In another embodiment, R ¹ is — (C _1-6 alkylene) -heterocycle.

In yet another embodiment, R ¹ is —CH ₂ -heterocycle.

In a further embodiment, R ¹ is —O— (C _1-6 alkylene) -heterocycle.

In another embodiment, R ¹ is —O—CH ₂ -heterocycle.

In another embodiment, R ¹ is — (C _1-6 alkylene) —N (R ⁴ ) _2, wherein each R ⁴ is independently —H or C _1-6 alkyl.

In another embodiment, R ¹ is — (C _1-6 alkylene) -NH (C _1-6 alkyl).

In another embodiment, R ¹ is — (C _1-6 alkylene) —N (R ⁴ ) _2, wherein both R ⁴ groups are — (C _1-6 alkylene)-(O—C _1-6 alkyl). ).

In embodiments, R ¹ is —CH ₂ —N (R ⁴ ) _2, wherein each R ⁴ is independently —H or C _1-6 alkyl.

In one embodiment, R ⁷ is —H.

In one embodiment, R ⁷ is -halo.

In one embodiment, R ⁷ is —O— (C _1-6 alkyl).

In another embodiment, R ⁷ is —O— (C _1-6 alkylene) -heterocycle.

In yet another embodiment, R ⁷ is —C (O) — (C _1-6 alkyl).

In another embodiment, R ⁷ is —O— (C _1-6 haloalkyl).

In one embodiment, R ⁸ is —H.

In another embodiment, R ⁹ is —H.

In yet another embodiment, R ⁸ is —H and R ⁹ is —H.

In a preferred embodiment, R ¹ is — (C _1-6 alkylene) -heterocycle and R ⁷ is —O— (C _1-6 alkyl).

In a more preferred embodiment, R ¹ is — (C _1-6 alkylene) -heterocycle, R ⁷ is —O— (C _1-6 alkyl), R ⁸ is —H, and R ⁹ is -H.

In another preferred embodiment, R ¹ is — (C _1-6 alkylene) —N (R ⁴ ) ₂ , R ⁷ is —O— (C _1-6 alkyl), and R ⁸ is —H. Yes, R ⁹ is —H.

Examples of indazole compounds having the formula (Ia) include the following compounds and isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrates thereof:

  The invention also targets multiple kinases in a composition comprising a therapeutically effective amount of an indazole compound having formula (Ia) and a pharmaceutically acceptable vehicle and diseases treatable by targeting multiple kinases, Also provided is use in the treatment of a disorder or condition.

  In a particularly preferred embodiment, the single drug is Compound 20 above, designated herein as “CC001”. See Examples 1 and 85.

In another embodiment, the invention is an indazole compound having formula (Ib), and isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrates thereof.

In the above formula,
R ¹ is —H, —C _1-6 alkyl, — (C _1-6 alkylene) -R ² or —O— (C _1-6 alkylene) -R ² ;
R ² is —C _1-6 alkyl, —C _1-6 alkoxy, —OH, —N (R ³ ) ₂ , —aryl, —heteroaryl, —heterocycle or —cycloalkyl;
Each R ³ is independently —H, —C _1-6 alkyl or —C _1-6 alkylene- (C _1-6 alkoxy);
R ⁴ represents —N (R ⁵ ) ₂ , —O—C _1-6 alkyl, —C (O) NH— (C _1-6 alkylene) _m -heterocycle, —C (O) -heterocycle, —C (O) NH- (C _1-6 alkylene) _m -heteroaryl, -C (O) -heteroaryl,-(C _1-6 alkylene) -cycloalkyl, -O- (C _1-6 alkylene) -N _{^{(R 5) 2, -O- (}} C 1-6 _{alkylene) m} - heterocycle, -O- _{(C 1-6 alkylene) m} - heteroaryl, -O- _{(C 1-6 alkylene) m} - cycloalkyl Or —O— (C _1-6 alkylene) _m —C (O) R ⁵ ;
Z is —CH— or —N—;
Each R ⁵ is independently —H or —C _1-6 alkyl;
m is 0 or 1.

  In one embodiment, -Z is -CH-.

In another embodiment, R ¹ is —H.

In yet another embodiment, R ¹ is —C _1-6 alkyl.

In another embodiment, R ¹ is — (C _1-6 alkylene) -heterocycle.

In yet another embodiment, R ¹ is —CH ₂ -heterocycle.

In a further embodiment, R ⁴ is —N (R ⁵ ) ₂ .

In a further embodiment, R ⁴ is —O— (C _1-6 alkylene) -heterocycle.

In a further embodiment, R ⁴ is —O—C _1-6 alkyl, preferably —OCH ₃ .

In another embodiment, R ⁴ is —O—CH ₂ -heterocycle.

In another embodiment, R ⁴ is —O— (CH ₂ ) ₂ -heterocycle.

In another embodiment, R ⁴ is —O— (CH ₂ ) ₃ -heterocycle.

In another embodiment, R ⁴ is —O— (C _1-6 alkylene) -heterocycle.

In a preferred embodiment, R ¹ is —H and R ⁴ is —O— (C _1-6 alkylene) -heterocycle.

In another preferred embodiment, R ¹ is —CH ₂ -heterocycle and R ⁴ is —O— (C _1-6 alkylene) -heterocycle.

In yet another preferred embodiment, R ¹ is —C _1-6 alkyl and R ⁴ is —O— (C _1-6 alkylene) -heterocycle.

In yet another preferred embodiment, Z is —CH—, R ¹ is — (C _1-6 alkylene) -R ² , R ² is —N (R ³ ) ₂ , and R ⁴ is — it is O-CH _3.

In yet another preferred embodiment, Z is —CH—, R ¹ is —C _1-6 alkyl, and R ⁴ is —O— (C ₂ alkylene) —N (R ⁵ ) ₂ .

Examples of indazole compounds having the formula (Ib) include the following compounds and isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrates thereof:

  The invention also targets multiple kinases in a composition comprising a therapeutically effective amount of an indazole compound having formula (Ib) and a pharmaceutically acceptable vehicle, and diseases treatable by targeting multiple kinases, Also provided is use in the treatment of a disorder or condition.

  In a particularly preferred embodiment, the single drug is Compound 52 above, designated herein as “CC002”. In another particularly preferred embodiment, the single drug is Compound 92 above, designated herein as “CC0004”. In a particularly preferred embodiment, the single drug is Compound 117 above, designated herein as “CC005”. See Examples 1 and 86-88.

In another embodiment, the invention treats a treatable disease, condition or disorder by targeting (eg, inhibiting) multiple protein kinases to target (eg, inhibit) multiple kinases. For the use of a single drug for. However, the single drug is 5- (5-cyclopentyl-1H- [1,2,4] triazol-3-yl) -3- (4-fluorophenyl-1H-indazole):

However, 3- (5- (1H-1,2,4-triazol-3-yl) (1H-indazol-3-yl) phenyl) -N-cyclopentylcarboxamide

not.

  In another embodiment, the methods of the invention involve targeting multiple kinases with a single drug that is a small (<1000 dalton) organic molecule that is not an intazole-containing compound. In another embodiment, the methods of the invention involve targeting multiple kinases with a single drug that is a nucleic acid (eg, aptamer) that modulates the activity of multiple protein kinases. In another embodiment, the methods of the invention include targeting multiple kinases with a single drug that is a peptide that modulates the activity of multiple kinases. These single drugs and other drugs described herein can be used in combination with each other. For example, a single drug that is an indazole-containing compound can be used with an indazole-containing compound, a single drug that is a polynucleotide or a polypeptide, or a combination thereof. A single drug that is a peptide can be used with a single drug, such as an indazole-containing compound, a non-indazole-containing compound, or a polynucleotide. In a specific embodiment, the methods of the invention include targeting with a plurality of protein kinase peptides, polynucleotides or non-indazole small molecules that are CDK kinase, Yes kinase, cSrc kinase, MEK kinase and Rsk kinase. In a more specific embodiment, the plurality of kinases described above consists of human CDK1, CDK2, Yes, MEK1, cSRC and Rsk1.

(4.3) Adjuvant treatment According to the present invention, treatment by administration of one or more single drugs may be combined with administration of one or more treatments. Such treatments include, but are not limited to, chemotherapy, radiation therapy, hormone therapy and / or biological therapy / immunotherapy.

(4.3.1) Cancer Adjunctive Treatment In certain embodiments, the methods of the invention involve the administration of one or more angiogenesis inhibitors with one or more single drugs. Examples of the angiogenesis inhibitor include angiostatin (plasminogen fragment), anti-angiogenic antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab, BMS-275291, cartilage-derived inhibitor ( CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), fibronectin fragment, Gro-β, halofuginone, heparinase, heparin hexasaccharide fragment, HMV833, Human chorionic gonadotropin (hCG), IM-862, interferon α / β / γ, interferon-inducible protein (IP-10), interleukin 12, kringle 5 (plasminogen fragment), marimastat, metallo Proteinase inhibitor (TIMP), 2-methoxyestradiol, MMI 270 (CGS 27023A), MoAb IMC-1C11, Neobastat, NM-3, Panzem, PI-88, placental ribonuclease inhibitor, plasminogen activator inhibitor , Platelet factor 4 (PF-4), purinomastert, prolactin 16 kD fragment, proliferin-related protein (PRP), PTK 787 / ZK 222594, retinoid, sorimastate, squalamine, SS 3304, SU 5416, SU 6668, SU 11248, tetrahydro Cortisol-S, tetrathiomolybdate, thalidomide, thrombospondin 1 (TSP-1), TNP-470, transforming growth factor β (TGF-β), bascrostatin, Examples include, but are not limited to, vasostatin (calreticulin fragment), ZD6126, ZD6474, farnesyltransferase inhibitor (FTI) and bisphosphonate.

Additional examples of anti-cancer agents that can be used in various embodiments of the present invention, including pharmaceutical compositions, dosage forms and kits of the present invention include acivicin, aclarubicin, acdazole hydrochloride, acronin, adzelsin, aldesleukin, altretamine, ambomycin, acetic acid Amethanetron, aminoglutethimide, amsacrine, anastrozole, anthromycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene, dimesylate bisnafide, bzelcine, bleomycin sulfate, brequinar sodium, bropyrimine, Busulfan, Kactinomycin, Carsterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin hydrochloride, Carzelsi , Cedefine gol, chlorambucil, cilolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, decarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexolmaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, docetaxel , Doxorubicin hydrochloride, droloxifene, droloxifene citrate, drmostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enroplatin, enpromate, epipropidine, epirubucin hydrochloride, elbrozol, esorubicin hydrochloride, estramustine, phosphoric acid Estramustine sodium, etanidazole, etoposide, etoposide phosphate Etoprine, Fadrozole hydrochloride, Fazarabine, Fenretinide, Floxlidine, Fludarabine phosphate, Fluorouracil, Fluorocitabine, Foskidone, Fostriecin sodium, Gemcitabine, Gemcitabine hydrochloride, Hydroxyurea, Idarubicin hydrochloride, Ifosfamide, Irmofosine, Interleukin II Leukin II or rIL2), interferon-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-Ia, interferon γ-Ib, iproplatin, irinotecan, lanreotide acetate, letrozole, leuprolide acetate , Riarosol hydrochloride, lometrexol sodium, lomustine, rosoxanthrone hydrochloride, Soprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogalyl, mercaptopurine, methotrexate, methotrexate sodium, methoprin, metredepa, mitidomid, mitocalcin, mitochromine, mitogylline, mitmarcin, mitomycin, mitomycin Mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogaramycin, ormaplatin, oxythran, paclitaxel, pegase pargase, peromycin, pentamustine, pepromycin sulfate, perphosphamide, pipbloman, piposulfan, pyroxanthrone hydrochloride, prikamycin, promestan, porfimel sodium , Porphyromycin, predoni Mustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurine, logretimide, saphingol, saphingol hydrochloride, semustine, simtrazen, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptoni Glin, streptozocin, throfenur, tarisomycin, tecogalane sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxylone, test lactone, thiapurine, thioguanine, thiotepa, thiazofurin, tirapazamine, tremifen citrate, trestron acetate acetate, triciribine phosphate, Trimetrexate, Trimethrexate glucuronate, Triptorelin, Tubrosol hydrochloride, Rasil mustard, uredepa, vapleotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinpidine sulfate, vinlicine sulfate, vinleucine sulfate, vinorelbine tartrate, vinrosidin sulfate, vinzolidine sulfate, borosol, zeniplatin, dinostatin, zorubicin hydrochloride Including, but not limited to. Other anti-cancer agents include 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclavicin, acylfulvene, adecipenol, adzelsin, aldesleukin, ALL-TK antagonist, altretamine, ambermustine, amidox, amifostine, aminolevulinic acid , Amrubicin, Amsacrine, Anagrelide, Anastrozole, Andrographolide, Angiogenesis inhibitor, Antagonist D, Antagonist G, Antarelics, Antidorpomorphic morphogenic protein-1, Antiandrogen, Prostate cancer, Antiestrogen, Antineopra Stone, antisense oligonucleotide, aphidicolin glycinate, apoptosis gene modulator, apoptosis regulator, aprinic acid, ara-CDP- L-PTBA, arginine deaminase, aslacrin, atamestan, atormistin, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, valanol, batimastat, BCR / ABL antagonist, benzochlorin, Benzoylstaurosporine, β-lactam derivatives, β-alletin, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, biselecin, breflate, bropirimine, butitanium, butionine Sulfoximine, calcipotriol, calphostin C, camptothecin derivative, canalipox IL-2, capecitabine, carboxamide- Mino-triazole, carboxamidotriazole, CaRest M3, CARN 700, cartilage-derived inhibitor, calzeresin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorlin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin , Cladribine, clomiphene analog, clotrimazole, chorismycin A, chorismycin B, combretastatin A4, combretastatin analog, conagenin, clambecidin 816, crisnatol, cryptophycin 8, cryptophicin A derivative, clasin A, cyclopen Tantraquinone, cycloplatam, cypemycin, cytarabine ocphosphate, cytolytic factor, cytostatin, dacliximab, deci Tabine, Dehydrodidemnin B, Deslorelin, Dexamethasone, Dexifosfamide, Dexrazoxane, Dexverapamil, Diaziquan, Didemnin B, Didox, Diethylnorspermine, Dihydro-5-azacytidine, Dihydrotaxol, 9-, Dioxamycin, Diphenylspirin Lomustine, docetaxel, docosanol, dolasetron, doxyfluridine, droloxifene, dronabinol, duomycin SA, ebselen, ecomustine, edelfocin, edrecolomab, eflornithine, elemen, emiteflu, epirubicin, epristeride, estramogen agonist, oestrogen agonist, estrogen agonist Etanidazole, etoposide phosphate, exemestane, fadrozole, Azalabine, fenretinide, filgrastim, finasteride, flavopiridol, fluasterastin, fluasterone, fludarabine, fluorodaunolnnisin hydrochloride, phorfenimex, formestane, hostriecin, hotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganilex inhibitor , Gemcitabine, glutathione inhibitor, hepsulfam, heregulin, hexamethylenebisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramanton, ilmofosin, ilomastert, imidazoacridone, imiquimod, immunostimulatory peptide, insulin-like growth factor 1 receptor inhibition Agent, interferon agonist, interferon, interleukin, iove Guan, iododoxorubicin, ipomeanol, 4-, iropract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jaspraquinolide, kahalalide F, lamellarin-N triacetate, lanreotide, reinamycin, lenograstim, lentinan sulfate, reptor Statins, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, riarosol, linear polyamine analogues, lipophilic disaccharide peptides, lipophilic platinum compounds, lysoclinamide 7, lobaplatin, lombricin, lometrexol, Lonidamine, rosoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysophy , Dissolved peptide, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitor, matrix metalloproteinase inhibitor, menogalil, melvalon, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirimostim, Inappropriate double-stranded RNA, mitoguazone, mitactol, mitomycin analog, mitonafide, mitoxin fibroblast growth factor-saporin, mitoxantrone, mofaroten, morglamostim, monoclonal antibody, human chorionic gonadotropin, monophosphoryl lipid A + myobacterium cell wall sk , Mopidamol, multidrug resistance gene inhibitor, treatment based on multiple tumor suppressor 1, mustard anticancer agent, mi Peroxide B, mycobacterial cell wall extract, myriapolone, N-acetyldinarine, N-substituted benzamide, nafarelin, nagres chip, naloxone + pentazocine, napabin, naphtherpine, nartograstim, nedaplatin, nemorubicin, neridronate, neutral endopeptidase , Nilutamide, nisamycin, nitric oxide modulator, nitric oxide antioxidant, nitrulline, O6-benzylguanine, octreotide, oxenone, oligonucleotide, onapristone, ondansetron, ondansetron, olacin, oral cytokine inducer, ormaplatin , Osaterone, Oxaliplatin, Oxaunomycin, Paclitaxel, Paclitaxel analog, Paclitaxel derivative, Parauamine, Palmitoyl Zoxin, pamidronic acid, panaxitol, panomiphene, parabactin, pazelliptin, pegase pargase, perdesin, pentozan polysulfate sodium, pentostatin, pentrozole, perflubron, perphosphamide, perillyl alcohol, phenazinomycin, phenylacetic acid, phosphatase inhibitor, picibanil , Pilocarpine hydrochloride, pirarubicin, pyritrexim, pracetin A, pracetin B, plasminogen activator inhibitor, platinum complex, platinum compound, platinum-triamine complex, porfimer sodium, porphyromycin, prednisone, propylbis-acridone , Prostaglandin J2, proteasome inhibitor, protein A-based immune modulator, protein kinase C inhibitor, tan Protein kinase C inhibitor, microalgal, protein tyrosine phosphatase inhibitor, purine nucleoside phosphorylase inhibitor, purpurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonist, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitor, ras inhibitor, ras-GAP inhibitor, demethylated retelliptin, rhenium Re 186 etidronate, lysoxin, ribozyme, RII retinamide, rogretimide, lohitskin, lomultide, rokinimex, rubiginone B1, ruboxil, saphingol, sinepineol , Sargramostim, Sdi 1 Mimetics, Semustine, Senescence Inducible inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single chain antigen binding protein, schizophyllan, sobuzoxane, sodium borocaptate, sodium phenylacetate, sorbelol, somatomedin binding protein, sonermin, spurfos acid , Spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipamide, stromelysin inhibitor, sulfinosine, overactive vasoactive intestinal peptide antagonist, sradista, suramin, swainsonine , Synthetic glycosaminoglycans, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalane sodium, tegafur, tellapyrylium, Lomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, salivlastine, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfacin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, chinethyl etiopurpurin, Tilapazamine, titanocene dichloride, topcentin, toremifene, totipotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, triciribine, trimethrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitor, tyrophostin, UBC inhibitor, Ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonist, bapreotide, variolin B, vector system Erythrocyte gene therapy Beraresoru, Beramin, verdins, verteporfin, vinorelbine, Binkisaruchin, Vitaxin, vorozole, Zanoteron, Zenipurachin, including but Jirasukorubu and zinostatin stimulating factor, but not limited to. Preferred additional anticancer agents are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods using thalidomide and topoisomerase inhibitors.

In a more specific embodiment, the invention also includes the administration of one or more single drugs in combination with the administration of one or more treatments. The aforementioned treatments include anticancer agents as disclosed in Table 1, but are not limited to these anticancer agents.

  The invention also encompasses the administration of one or more single drugs in combination with radiation therapy including using X-rays, gamma rays and other radiation sources to destroy cancer cells. In a preferred embodiment, the radiation therapy is administered as external beam radiation therapy or teleradiation therapy that applies radiation from a remote source. In other preferred embodiments, radiation therapy is administered as an internal therapy or brachytherapy in which the radiation source is placed in the body near the cancer cells or tumor mass.

  Cancer therapies and their doses, routes of administration and recommended dosages are known in the art and are described in literature such as the Physician's Desk Reference (56th edition, 2002).

(4.3.2) Inflammatory adjuvant treatment To treat inflammation and inflammation-related conditions, one or more single drugs may be administered in combination with one or more compounds having anti-inflammatory activity. Accordingly, treatment with a single drug in various embodiments includes, for example, steroidal anti-inflammatory agents, non-storoidal anti-inflammatory agents (NSAIDs), Benadryl®, IL-9 antagonists, antihistamines, sympathomimetics, Glucocorticoids, corticosteroids, β-adrenergic drugs (epinephrine and isoproterenol), theophylline, anticholinergics (eg, atropine and ipratropium bromide), leukotriene inhibitors, immunotherapy (eg, allergen repeated long term Injection, short-term desensitization, toxic immunotherapy, effective amount of one or more anti-IgE antibodies and / or one or more mast cell modulators (eg, mast cell protease inhibitor, stem cell factor (c-kit ligand) inhibition) Combined with treatments using pharmacological agents and c-kit receptor inhibitors) It can be.

(4.4) Dose and Administration The present invention relates to the administration of one or more compounds (also referred to as single drugs) that act to simultaneously target two or more kinases or genes encoding them.

  In a preferred embodiment, the single drug is a pharmaceutical composition. The composition comprises a prophylactically or therapeutically effective amount of one or more single drugs and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” is approved by a federal or state regulatory authority for use on animals, particularly humans, or is recognized by the US Pharmacopeia or other generally accepted. Means it is listed in the pharmacopoeia. The term “carrier” refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle that is administered with a therapeutic agent. Such pharmaceutical carriers are sterile liquids, such as water and oils (of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol , Water, ethanol and the like. If desired, the composition may also contain minor amounts of wetting / emulsifying agents or pH buffering agents. The composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral compositions can be formulated with common carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E. W. Martin's “Remington's Pharmaceutical Sciences”. The composition comprises a prophylactically or therapeutically effective amount (preferably in purified form) of a prophylactic or therapeutic agent together with a suitable amount of carrier for proper administration to a patient. The formulation must be compatible with the mode of administration. In a preferred embodiment, the pharmaceutical composition is sterile and has a form suitable for administration to a subject, preferably an animal subject, more preferably a mammalian subject, most preferably a human subject.

  In a specific embodiment, it may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment. This can be achieved by way of example and not limitation, using local injection, injection or an implant. The implant is made of a porous, non-porous or gelatinous material or fiber, including a membrane such as a silastic membrane. Preferably, care should be taken to use materials that do not absorb the prophylactic or therapeutic agent when administering one or more prophylactic or therapeutic agents.

  In another embodiment, a single drug can be delivered in vesicles, particularly liposomes (Langer, Science, 249: 1527-1553 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Ber Edited by Fiddles, published in Liss (1989), New York, p. 353-365; see Lopez-Berstein, p. 317-327 above; see above for an overview).

  In yet another embodiment, a single drug can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used for controlled release or sustained release (Langer, supra, CRC Crit. Ref. Biomed. Eng., 14:20 (1987); Buchwald et al., Surgary, 88: supra. 507 (1980); see Saudek et al., N, Engl. J. Med., 321: 574 (1989)). In another embodiment, polymeric materials can be used to control release or sustained release of antibodies or fragments thereof of the invention (eg, CRC Press located in Medical Applications of Controlled Release, Langer and Wise, Boca Raton, Florida). (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, edited by Smolen and Ball, published by Wiley (1984), New York; Ranger and Peppas, J. Macromol. (1983); Levy et al., Science, 228: 190 (198). 5); During et al., Ann. Neurol., 25: 351 (1989); Howard et al., J. Neurosurg., 71: 105 (1989); US Pat. No. 5,679,377, US Pat. 597, US Patent No. 5,912,015, US Patent No. 5,989,463, US Patent No. 5,128,326, International Patent Application Publication No. 99/15154 and International Patent Application Publication No. 99 / See also 20253). Examples of polymers used in sustained release formulations include poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid) , Polyglycoside (PLG), polyanhydride, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-co-glycolide) (PLGA) And polyorthoesters, but are not limited thereto. In a preferred embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile and biodegradable. In yet another embodiment, a controlled release or sustained release system can be placed near the therapeutic target, i.e., in the lung, and thus only a portion of the systemic dose is required (e.g., Goodson, Medical Applications of supra). Controlled Release, 2: 115-138 (1984)).

  Controlled release systems are discussed in a paper by Langer (Science, 249: 1527-1533 (1990)). Techniques known to those skilled in the art can be used to make sustained release formulations comprising one or more antibodies of the invention or fragments thereof. See, for example, US Pat. No. 4,526,938; International Patent Application Publication No. 91/05548; International Patent Application Publication No. 96/20698; Ning et al., “Infrared Radioimmunity of a Human, all of which are incorporated herein by reference. Colon Cancer Xenograft Using a Sustained-Release Gel ", Radiotherapy & Oncology, 39: 179-189 (1996); Song et al.," Antibody Mediated Lung Targeting of Long-Circulating Emulsions ", PDA Journal of Pharmaceutical Science & Technolog , 50: 372-397 (1995); Cleek et al., "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Proc. Int'l. Symp. Control. Rel. Bioact. Mater. , 24: 853-854 (1997); and Lam et al., “Microencapsulation of Recombinant Humanized Monoclonal Antibodies for Local Delivery”, Proc. Int'l. Symp. Control Res. Bioact. Mater. 24: 759-760 (1997).

  In a specific embodiment where the single drug of the invention is one or more nucleic acid molecules encoding one or more prophylactic or therapeutic agents, the nucleic acid is constructed in vivo as part of a suitable nucleic acid expression vector, and cells For example, use of retroviral vectors (US Pat. No. 4,980,286); direct injection; use of microparticle bombardment (eg, gene gun; Biolistic, Dupont); lipid or cell surface receptor or transfection Coating with a substance; by administration linked to a homeobox-like peptide known to enter the nucleus (see, for example, Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-1868 (1991)) It can be administered to promote the expression of the encoded prophylactic or therapeutic agent. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination.

  The pharmaceutical composition of the invention is formulated to be compatible with the intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), intranasal, transdermal (topical), transmucosal and rectal administration. In a specific embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. In a preferred embodiment, the pharmaceutical composition is manufactured according to routine procedures for subcutaneous administration to humans. Typically, an intravenous composition is a solution in a sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizing agent and a local anesthetic (eg, lignocaine) to ease pain at the site of the injection.

  If a single drug is to be administered topically, it may be formulated, for example, in an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or other form known to those skilled in the art. obtain. See, for example, Remington's Pharmaceutical Sciences and Induction to Pharmaceutical Dosage Forms, 4th edition, Lea & Febiger, Philadelphia, PA (1985). For topical dosage forms other than sprays, a viscous to semi-solid or solid form that is compatible with topical application and preferably comprises a carrier or dynamic excipient (preferably higher than water) or one or more excipients. used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, waxes and the like. The formulation may be sterilized if desired, but may also be mixed with auxiliary agents such as preservatives, stabilizers, wetting agents, buffers or salts to affect various properties (eg, osmotic pressure). For other suitable topical dosage forms, the active ingredient is preferably packaged in a mixture or squeeze bottle with compressed volatiles (eg, a gas propellant such as freon) in combination with a solid or liquid inert carrier. Sprayable aerosol formulations are included. If desired, humectants or wetting agents may be incorporated into the pharmaceutical compositions and dosage forms. Examples of additional ingredients are known in the art.

  Where the composition of the present invention is to be administered intranasally, the composition can be formulated in aerosol form, spray, mist or droplet form. In particular, a prophylactic or therapeutic agent for use in accordance with the present invention advantageously uses a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). It can be delivered in the form of an aerosol spray from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the single dose can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, such as gelatin capsules and cartridges for use in an inhaler or infuser, may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

  When the composition of the present invention is to be administered orally, the composition can be formulated in the form of tablets, capsules, cachets, gel caps, solutions, suspensions and the like. Tablets or capsules are pharmaceutically acceptable excipients such as binders (eg pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate) Lubricants (eg, magnesium stearate, talc or silica); disintegrants (eg, potato starch, sodium starch glycolate) or wetting agents (eg, sodium lauryl sulfate) can be used by conventional means. Tablets may be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or can be provided as a dry powder configured with water or other suitable vehicle prior to use. Liquid formulations are pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (eg lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters) , Ethyl alcohol or fractionated vegetable oil) and a preservative (eg, methyl or propyl p-hydroxybenzoate, or sorbic acid) can be prepared by conventional means. The formulations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated for sustained, controlled or sustained release of prophylactic or therapeutic agents.

  The compositions of the invention can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Injectable preparations may be provided in single dosage forms, such as ampoules or multi-dose containers, with a preservative added. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

  The compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, eg containing common suppository bases such as cocoa butter or other glycerides.

  In addition to the formulations described above, the compositions of the present invention can also be formulated as a depot formulation. Long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) and also by intramuscular injection. Thus, for example, the composition is formulated using a suitable polymer or hydrophobic material (eg, as an acceptable emulsion in oil) or ion exchange resin, or as a poorly soluble derivative (eg, a poorly soluble salt). Can be done.

  The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like; and cations such as sodium, potassium, ammonium, calcium, Salts formed using cations derived from iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like are included.

  In general, the components of the compositions of the present invention are in the form of a single dosage form, for example a lyophilized powder or anhydrous concentrate in a closed container (eg an ampoule or sachet) indicating the amount of the active ingredient Are supplied as separate or mixed. Where the composition is to be administered by infusion, it can be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be prepared so that the ingredients are mixed prior to administration.

  In particular, according to the present invention, one or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are contained in a sealed container (eg, an ampoule or sachet) indicating the amount of a single drug. In one embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the present invention is provided as a dry sterilized lyophilized powder or anhydrous concentrate in a sealed container, eg, water for administration to a subject. Or it can be restored to an appropriate concentration with saline solution. One or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are preferably at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 35 mg, Delivered in a single dose of 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The prophylactic or therapeutic agent or pharmaceutical composition of the present invention must be stored at 2-8 ° C. in the original sealed container, and the prophylactic or therapeutic agent or pharmaceutical composition of the present invention is preferably within one week after restoration, preferably It must be administered within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours or within 1 hour. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are supplied in liquid form in a sealed container indicating the amount and concentration of a single drug. The liquid form composition to be administered is preferably at least 0.25 mg / ml, more preferably at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml in a sealed container, Provided at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml or at least 100 mg / ml. The liquid form must be stored at 2-8 ° C. in the original sealed container.

  If desired, a single drug can be provided in the form of a pack or dispenser device that can contain one or more single dosage forms containing the active ingredients. The pack may be composed of metal or plastic foil, such as a blister pack. The pack or dispenser device described above may be accompanied by instructions for administration.

Identification of single drug kinase inhibitors Six compounds (putative single drugs) were tested against 50 kinases. The results are shown in Table 2. The numbers for each kinase indicate the% of control kinase activity in the presence of the specified drug at a concentration of 3 μM.

  Compounds CC001, CC002 and CC004 were most active against the kinases tested and inhibited each kinase activity of 16, 29 and 16 kinases listed in Table 2, respectively, by more than 95% compared to non-compound controls. CC004 showed good inhibitory activity against CDK1, CDK2, MEK1 and Rsk1, and slightly lower inhibitory activity against the Src family kinases cSrc and Yes. CC006 (5- (5-cyclopentyl-1H- [1,2,4] triazol-3-yl) -3- (4-fluoro-phenyl) -1H-indazole) and CC007 (3- (5- (1H- 1,2,4-triazol-3-yl) (1H-indazol-3-yl)) phenyl-N-cyclopentylcarboxamide) was included as a control.

In vitro effect of a single drug capable of targeting multiple kinases or kinase pathways CC001 is a low molecular weight mixed kinase inhibitor (MK1) with strong in vitro inhibitory activity against a wide range of kinases. CC001 inhibited the activation of kinase and its downstream target in HCT-116 colon cancer cells in a concentration-dependent manner. CC001 also showed strong antiproliferative activity against a wide range of cancer cell lines including non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, head and neck cancer, breast cancer and ovarian cancer. In either case, the IC ₅₀ value is in the nanomolar range. In an in vitro combination study using standard chemotherapeutic agents such as taxol and novel signaling inhibitors, CC001 showed additive and / or synergistic antiproliferative activity.

  CC001 has shown a cancer suppressive effect in an in vivo cancer model. CC001 with a concentration of 10-20 mg / kg was administered twice daily to treat SCID mice with tumors in the colon and lung. CC001 as a single drug suppressed tumor growth in a dose-dependent manner (T / C ratio: 40-60%).

  A similar study combining CC001 with the maximum tolerated taxotere (an anticancer compound related to taxol) was performed using the NCI-H460 NSCLC xenograft model. In this experiment, a T / C ratio of 16-28% was observed. No obvious additional toxicity that was evident with weight loss was observed. An additive in vivo effect was also observed with camptosar, an anticancer drug commonly used for colorectal cancer, with a T / C ratio of 15%.

In one experiment, female CB. The right hind limb of 17 SCID mice was inoculated subcutaneously with 2 × 10 ⁶ HCT-116 cells. After 8 days, mice with tumors usually in the size of 75-125 mm ³ were selected and randomly assigned to groups. Treatment began on day 8 and continued throughout the study. All compounds were i.v. in a volume of 5 ml / kg in vehicle NPS. p. Administered. CC001 is 20 mg / kg b. i. d. Administered. Camptosar is i. p. q4d was administered. Tumor volume was measured twice a week using a caliper. Values are mean ± SEM. Statistical analysis was performed using ANOVA. As shown in FIG. 1 and Table 3, treatment with CC001 greatly reduced tumor size during the experiment (P <0.001 compared to vehicle control).

In a related experiment, two compounds designated CC002 and CC003 were tested. CC003 is naphthalene-containing indole. Female CB. The right hind limb of 17 SCID mice was inoculated subcutaneously with 2 × 10 ⁶ HCT-116 cells. After 8 days, mice with tumors usually in the size of 75-125 mm ³ were selected and randomly assigned to groups. Treatment began on day 8 and continued throughout the study. All compounds were i.v. in a volume of 5 ml / kg in vehicle NPS. p. Administered. CC002 and CC003 are 20 mg / kg and the first 4 days (day 8-11) are b. i. d. Administration was followed by qd. Camptosar was administered at a dose of 25 mg / kg q4d. Tumor volume was measured twice a week using a caliper. Values are mean ± SEM. Statistical analysis was performed using ANOVA. As shown in FIG. 2 and Table 4, treatment with CC002 or CC003 reduced tumor volume during the experiment (P <0.001 compared to vehicle control).

Compound C001 is highly effective when administered with a second compound. Female CB. The right hind limb of 17 SCID mice is inoculated subcutaneously with 2 × 10 ⁶ HCT-116 cells. After 8 days, mice with tumors usually in the size of 75-125 mm ³ were selected and randomly assigned to groups. Treatment began on day 8 and continued throughout the study. All compounds were i.v. in a volume of 5 ml / kg in vehicle NPS. p. Administered. CC001 is 20 mg / kg b. i. d. Administered. Camptosar is i. p. q4d was administered. Tumor volume was measured twice a week using a caliper. Values are mean ± SEM. Statistical analysis was performed using ANOVA. As shown in FIG. 3 and Table 5, administration of CC001 at 20 mg / kg with 2.5 mg / kg camptosar showed an effect equivalent to administration of only 10 mg / kg camptosar on tumor growth (vehicle control). Compared to P <0.001).

In Vivo Assay to Evaluate Candidate Single Drug A single drug can be confirmed or ultimately identified using various in vivo models. Typically, candidate single drugs are first identified on an in vitro screen and confirmed in an in vitro model for the particular pathological condition studied.

  First, a suitable in vitro model is selected. For example, a mouse tumor model can be used for many types of cancer, including cancers with specific metastatic patterns, and serves as an information center for information about available mouse tumor models (MTBOP). Can be selected from known sources such as MTBOP is available on tumor. informations. jax. org / FMPro? -Db = TumorInstance & -format = mtdp. Available at html & -view. Mouse inflammation and diabetes models are available, for example, under the name Jax Mice & Services from The Jackson Laboratory (located in Bar Harbor, Maine). Each of jax. org / jaxmice and jammice. jax. org / jaxmicedb / html / sbmodel — 7. See shml. Other disease models in mice or other mammals can also be used. For example, mouse, hamster or rat models of arthritis and diabetes are known.

  As a second example, the anti-inflammatory activity of a putative single drug can be assessed using a carrageenan-induced arthritis rat model. Carrageenan-induced arthritis has been used in rabbits, dogs and pigs in studies of chronic arthritis or inflammation. Quantitative histomorphometric evaluation is used to examine therapeutic effects. The method using the carrageenan-induced arthritis model is described in P.A. Hansra, “Carrageenan-Induced Arthritis in the Rat”, Information, 24 (2): 141-155 (2000). Also commonly used are zymosan-induced inflammation animal models known in the art and published. The anti-inflammatory activity of a putative single drug has shown that C. carrageenan-induced limb edema is inhibited in rats. A. Winter et al., “Carrageenan-Induced Edem in Hind Paw of the Rats as an Assay for Anti-Inflammatory Drugs”, Proc. Soc. Exp. Biol. Med. 111: 544-547 (1962). This assay has been used as a primary in vivo screen for the anti-inflammatory activity of many NSAIDs and is considered expected to have an effect on humans. The anti-inflammatory activity of the putative single drug tested is expressed as the percent inhibition of hindlimb weight increase compared to the control group receiving vehicle.

Animal models of asthma can also be used to assess the effects of putative single drugs. For example,
Cohn et al. Exp. Med. 186: 1737-1747 (1997).

  Animal models of autoimmune diseases can also be used to assess the effects of putative single drugs. Animal models for autoimmune diseases such as type 1 diabetes, thyroid autoimmunity, systemic lupus erythematosus and glomerulonephritis have also been developed (Flanders et al., Autoimmunity, 29: 235-246 (1999); Krogh et al., Biochimie, 81). : 511-515 (1999); Foster, Semin. Nephrol., 19: 12-24 (1999)).

In vivo models can also be created. For example, a tumor model can be created by administering a specific tumor model to a mouse. As an example, a colon cancer model can be constructed as follows. MCA26 is a colon cancer tumor cell line chemically induced in BALB / c mice (Corbett et al., 1975). Metastatic colon cancer is induced by transplanting approximately 7 × 10 ⁴ MCA26 cells into the left lobe of the liver of 8-10 week old female BALB / c mice (Taconic). On day 7, mice with a 5 × 5 mm ² size tumor are selected for administration of the putative single drug. Alternatively, the cells may be administered intraperitoneally or via the tail vein.

  Once a model is selected, a specific kinase is identified with possible targeting. For example, in the case of a tumor model, an appropriate kinase for the target may be known to be involved in cell cycle control, adhesion signaling or angiogenesis. Kinases with unknown or poorly characterized roles in specific pathological conditions can also be tested. In this case, however, inhibition or modulation of kinase activity must be correlated with an appropriate desired response (eg, decreased proliferation).

  The particular compound (ie, a putative single drug) is then examined for the ability to affect the morbid state under study. A single drug to be tested is administered to a test animal and the physiological response of the pathological condition is compared to a control (usually an animal that has received only the carrier for the single drug). The criteria for effect depend on the pathological condition being studied. For example, typical efficacy criteria for tumor models are obvious suppression of weight loss, reduction or suppression of tumor size expansion, suppression of metastasis, reduction of abnormal cell count in tissue pieces, and the like. Criteria are subjective (eg, improved appearance or appearance of health) or objective (difference between control and experimental groups is usually statistically determined). Kinase activity and changes in activity can be examined by taking blood or tissue samples by common means and testing kinase activity using one or more of the assays exemplified in Examples 4-83.

  A single drug is identified as a drug that affects more than one kinase in the in vivo assay described above and has at least one beneficial effect on the pathological condition under study.

  Examples 4-83: Assays for Measuring Candidate Activity: The following assays can be used to assess kinase activity. The assay is modified by adding the compound to be tested for kinase inhibition or modulation activity under experimental conditions. These examples of assays are not meant to be exclusive. The activity of various kinases can be assessed in other ways.

JNK2 assay 20% DMSO / 80% Add 50 ng His6-JNK2 (30 μl) in the same dilution buffer to the test compound (10 μl) in dilution buffer. The dilution buffer is 20 mM HEPES (pH 7.6) in water, 0.1 mM EDTA, 2.5 mM magnesium chloride, 0.004% Triton x100, 2 μg / ml leupeptin, 20 mM glycerophosphate, 0.1 mM sodium valerate and 2 mM. It is composed of DTT. Preincubate the mixture at room temperature for 30 minutes. Assay buffer consisting of 20 mM HEPES (pH 7.6) in water, 50 mM sodium chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mM DTT, 25 mM PNPP, 0.05% Triton x100, 11 μM ATP and 0.5 μCi γ- ³² P ATP 10 μg of GST-c-Jun (1-79) (60 μl) in solution is added and the reaction is allowed to proceed for 1 hour at room temperature. C-Jun phosphorylation is stopped by adding 12.5% trichloroacetic acid (150 μl). After 30 minutes, the precipitate is collected on a filter plate, diluted with scintillation fluid (50 μl) and quantified by a counter. IC ₅₀ values are calculated as the concentration of test compound at which c-Jun phosphorylation is reduced to 50% of the control value. Preferred compounds of the invention have an IC ₅₀ value of 0.01-10 μM in this assay.

JNK3 Assay 20% DMSO / 80% Add 50 ng His6-JNK3 (30 μl) in the same dilution buffer to the test compound (10 μl) in dilution buffer. The dilution buffer is 20 mM HEPES (pH 7.6) in water, 0.1 mM EDTA, 2.5 mM magnesium chloride, 0.004% Triton x100, 2 μg / ml leupeptin, 20 mM glycerophosphate, 0.1 mM sodium valerate and 2 mM. It is composed of DTT. Preincubate the mixture at room temperature for 30 minutes. Assay buffer consisting of 20 mM HEPES (pH 7.6) in water, 50 mM sodium chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mM DTT, 25 mM PNPP, 0.05% Triton x100, 11 μM ATP and 0.5 μCi γ- ³² P ATP 10 μg of GST-c-Jun (1-79) (60 μl) in solution is added and the reaction is allowed to proceed for 1 hour at room temperature. C-Jun phosphorylation is stopped by adding 12.5% trichloroacetic acid (150 μl). After 30 minutes, the precipitate is collected on a filter plate, diluted with scintillation fluid (50 μl) and quantified by a counter. IC ₅₀ values are calculated as the concentration of test compound at which c-Jun phosphorylation is reduced to 50% of the control value. Preferred compounds of the invention have an IC ₅₀ value of 0.01-10 μM in this assay.

Junkat T cell IL-2 production assay Junkat T cells (clone E6-1) were purchased from American Type Culture Collection and received 2 mM L-glutamine (Mediatech), 10% fetal calf serum (Hyclone) and penicillin / streptomycin. Maintain in growth medium consisting of RPMI 1640 containing. All cells are cultured at 37 ° C. in 95% air and 5% CO ₂ . Cells are plated at a density of 0.2 × 10 ⁶ cells / well with 200 μl medium. Compound stock (20 mM) is diluted in growth medium and added to each well as a 10 × concentrated solution in a volume of 25 μl, mixed and pre-incubated with cells for 30 minutes. The compound vehicle (dimethyl sulfoxide) is maintained at a final concentration of 0.5% in all samples. After 30 minutes, cells are activated with PHA (phorbol myristate acetate; final concentration 50 μg / ml) and PHA (phytohemagglutinin; final concentration 2 μg / ml). PMA and PHA are added as a 10 × concentrated solution in growth medium and added in a volume of 25 μl / well. The cell plate is incubated for 10 hours. The cells are pelleted by centrifugation, the medium is removed and stored at -20 ° C. Media aliquots are analyzed by sandwich ELSA for the presence of IL-2 according to the manufacturer's (Endogen) instructions. IC ₅₀ values are calculated as the concentration of test compound at which IL-2 production is reduced to 50% of the control value. Preferred compounds of the invention have an IC ₅₀ value of 0.01-10 μM in this assay.

Rat In Vivo LPS Induced TNF-α Production Assay Seven week old male CD rats purchased from Charles River Laboratories are acclimated for one week prior to use. The outer tail vein is cannulated percutaneously using a 22 gauge or larger catheter with light isoflurane anesthesia. Rats are administered test compounds 15-180 minutes prior to injection of 0.05 mg / kg LPS (E. coli 0.55: BS) by intravenous infusion via the tail vein catheter or oral irrigation. The catheter is washed with 2.5 ml / kg of saline for injection. Blood is collected by cardiac puncture 90 minutes after LPS challenge. Plasma is made using lithium heparin separators and frozen at -80 ° C until analysis. TNF-α levels are measured using a rat-specific TNF-α ELISA kit (Biosource). The ED ₅₀ value is calculated as the dose of test compound that reduces TNF-α production to 50% of the control value. Preferred compounds of the invention have an EC ₅₀ value of 1-30 mg / kg in this assay.

CDC kinase activity assay cyclin-dependent kinase activity can be examined by quantifying the ^{[32 P]} ATP or ^{[33 P]} of an enzyme catalyst into a protein substrate of radioactive phosphate from ATP time-dependent uptake. Unless otherwise stated, the assay was performed in a total volume of 50 μl in a 96-well plate, 10 mM HERPES (N- [2-hydroxyethyl] piperazine-N ′-[2-ethanesulfonic acid]) (pH 7) per reaction. .4) 10 mM MgCl ₂ , 25 μM adenosine triphosphate (ATP), 1 mg / ml ovalbumin, 5 μg / ml leupeptin, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 0.1 mM sodium valerate, 1 mM sodium fluoride, 2.5 mM ethylene glycol bis (β-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid (EGTA), 2% (v / v) dimethyl sulfoxide and 0.03-0.4 μCi [ ³² / Performed in the presence of ³³ P] ATP (EDTA). The reaction is initiated with the appropriate enzyme, incubated at 30 ° C., and after 20 minutes the reaction is stopped by adding ethylenediaminetetraacetic acid (EDTA) to 250 mM. The phosphorylated substrate is then captured on a nitrocellulose or phosphocellulose membrane using a 96-well filtration manifold, and unincorporated radioactivity is removed by repeated washing with 0.85% phosphoric acid. Radioactivity is quantified by exposing the dried membrane to a phosphorimager.

  Apparent Ki values were determined by assaying enzyme activity in the presence of different concentrations of inhibitor compounds and subtracting the background radioactivity measured in the absence of enzyme. Inhibition data is fit to the formula for competitive inhibition using Kaleidagraph (Synergy Software) or to the formula for competitive tight binding inhibition using the software KineTic (BioKin, Ltd).

Inhibition of CDK4 / cyclin D retinoblastoma kinase activity A complex of human CDK4 and cyclin D3 or a genetically shortened (1-264) cyclin D3 complex with human CDK4 is expressed in a corresponding baculovirus expression vector (Meijer And Kim, “Chemical Inhibitors of Cyclin-Dependent Kinases”, Methods in Enzymol., 283: 113-128 (1997)), are purified using common biochemical chromatography techniques. The enzyme complex (5 or 50 nM) is assayed using 0.3-0.5 μg of purified recombinant retinoblastoma protein fragment (Rb) as substrate. The engineered Rb fragment (residues 386-928 of the native retinoblastoma protein; 62.3 kDa) contains most of the phosphorylation sites present in the native 106 kDa protein and six to facilitate purification. Contains a tag for histidine residues. Phosphorylated Rb substrate is captured by microfiltration on a nitrocellulose membrane and quantified using a phosphorimager as described above. For the measurement of tight junction binders, the concentration of enzyme complex is reduced to 5 nM and the assay period is extended to 60 minutes where the time dependence of product formation is linear.

Inhibition of CDK2 / cyclin A retinoblastoma kinase activity CDK2 was prepared according to Rosenblatt et al., “Purification and Crystallization of Human Cyclin-dependent Kinase 2”, J. Biol. Mol. Biol. , 230: 1317-1319 (1993). Cyclin A is purified from E. coli cells expressing full-length recombinant cyclin A, and a shortened cyclin A construct is made by limited proteolysis, and Jeffrey et al. purification "as described in complex", Nature, 376: 313-320 (July 27, 1995). A complex of CDK2 and proteolytic cyclin A is made and purified by gel filtration. The substrate for this assay is the same Rb substrate fragment used in the CDK4 assay, and the CDK2 / cyclin A and CDK4 / cyclin D3 assay methods are essentially the same except that CDK2 is present at 150 nM or 5 nM. K _i values are measured as described above.

VEGF Activity Assay Stimulation of cell proliferation by growth factors such as VEGF is dependent on induction of autophosphorylation of each receptor tyrosine kinase. Thus, the ability of protein kinase inhibitors to block cell growth induced by these growth factors is directly correlated to the ability to block receptor autophosphorylation. The following constructs are used to determine the protein kinase inhibitory activity of a compound.

VEGF-R2 construct for assay: This construct tests the ability of a test compound to inhibit tyrosine kinase activity. A cytosolic domain construct (VEGF-R2Δ50) of human vascular endothelial growth factor receptor 2 (VEGF-R2) lacking 68 central 50 residues of the kinase insert domain is expressed in a baculovirus / insect cell line . Of the 1356 residues of full-length VEGF-R2, the VEGF-R2Δ50 construct contains residues 806-939 and 990-1171, with one point mutation (E990V) in the kinase insert domain compared to wild-type VEGF-R2. ). Autophosphorylation of the purified construct is achieved by incubating the enzyme at 4 μM in 100 mM Hepes containing 5% glycerol and 5 mM DTT (pH 7.5) in the presence of 3 mM ATP and 50 mM MgCl ₂ at 4 ° C. for 2 hours. carry out. After autophosphorylation, this construct was found to have essentially the same catalytic activity as the wild-type autophosphorylated kinase domain construct. See Parast et al., Biochemistry, 37: 16788-16801 (1998).

  CHK1 construct for assay: Full-length human CHK1 with a His tag at the C-terminus (FL-CHK1) is expressed using a baculovirus / insect cell system. It contains 6 histidine residues (6 × His-tag) at the C-terminus of 476 amino acids human CHK1. The protein is purified by conventional chromatographic techniques.

VEGF-R2 assay-binding spectrophotometric (FLVK-P) assay: ADP production from ATP with phosphoryl transfer, oxidation of NADH with phosphoenolpyruvate (PEP) and pyruvate kinase (PK) and lactate dehydrokinase Binding to a system with (LDH). The oxidation of NADH can be monitored by examining the decrease in absorbance at 340 nm (ε ₃₄₀ = 6.22 cm ⁻¹ mM ⁻¹ ) using a Beckman DU 650 spectrophotometer. The assay conditions for phosphorylated VEGF-R2Δ50 (shown as FLVK-P in the table below) are 1 mM PEP in 200 mM Hepes (pH 7.5), 250 μM NADH, 50 units / ml LDH, 20 units / ml PK, 5 mM DTT, 5.1 mM poly (E ₄ Y ₁ ), 1 mM ATP and 25 mM MgCl ₂ . Assay conditions for non-phosphorylated VEGF-R2Δ50 (shown as FLVK in the table below) are 1 mM PEP, 250 μM NADH, 50 units / ml LDH, 20 units / ml PK, 5 mM DTT, 20 mM in 200 mM Hepes (pH 7.5). Poly (E ₄ Y ₁ ), 3 mM ATP, 60 mM MgCl ₂ and 2 mM MnCl ₂ . The assay is started with 5-40 nM enzyme. Ki values are determined by measuring enzyme activity in the presence of different concentrations of test compound. Data is analyzed using enzyme kinetics and Kaleidagraph software.

Production of ADP from ATP with phosphoryl transfer to the CHK1 assay synthetic substrate peptide Syntide2 (PLARTLSVAGLPGKK; SEQ ID NO: 1) is converted to phosphoenolpyruvate (PEP) via the action of pyruvate kinase (PK) and lactate dehydrogenase (LDH). ) To oxidize NADH. The oxidation of NADH can be monitored by examining the decrease in absorbance at 340 nm (ε340 = 6.22 cm ⁻¹ mM ⁻¹ ) using an HP8452 spectrophotometer. A typical reaction solution is 4 mM PEP, 0.15 mM NADH, 28 units / ml LDH, 16 units / ml PK, 3 mM DTT, 0.125 mM Syntide-2 in 50 mM Tris (pH 7.5) and 400 mM NaCl. Contains 0.15 mM ATP, 25 mM MgCl ₂ . The assay is started with 10 nM FL-CHK1. Ki values are determined by measuring initial enzyme activity in the presence of different concentrations of test compound. Data is analyzed using enzyme kinetics and Kaleidagraph software.

Inhibition of phosphorylated FGF receptor and LCK tyrosine kinase activity The cloning, expression and purification of the cytosolic domain (amino acids 456-766) of FGFR1 tyrosine kinase containing three amino acid substitutions (L457V, C488A and C84S) was described in M.M. Mohammadi, J .; Schlessinger and S.M. R. Hubbard, Cell, 86: 577-587 (1996). This domain is expressed in Sf9 insect cells using baculovirus vectors and the protein is purified using conventional techniques. LCK tyrosine kinase is expressed in insect cells as an N-terminal deletion from amino acid 223 to the end of the amino acid 509 protein. The N-terminus of the protein also had two amino acid substitutions P223M and c224D. The kinase is purified using conventional chromatographic methods.

Tyrosine kinase activity binds to the pyruvate kinase-catalyzed transfer of phosphate from phosphoenolpyruvate to ADP to form phosphorylated poly (Glu, Tyr; 4: 1) substrate and ADP to form and regenerate ATP Can be measured using a coupled continuous spectrophotometric assay. Pyruvate production is coupled to lactate dehydrogenase-catalyzed reduction of pyruvate, which forms lactate and at the same time converts NADH to NAD ⁺ . Loss of NADH is monitored by measuring absorbance at 340 nm (see, eg, Technikova-Dobrova et al., “Spectophotometric determination of functional 72 et al., 69, 72 liters of protein kinetics of protein kinases. ). Enzyme activity, 200mM HEPES (pH7.5), 2mM _{phosphoenolpyruvate, 0.3mM NADH, 20mM MgCl 2,} 100μM ATP, 5mM DTT, when the P-FCF or P-LCK assays 5.1 or 25mM poly ( Glu, Tyr) 4: 1, and 15 units / ml each in the presence of pyruvate kinase and lactate dehydrogenase. Phosphorylated FGF receptor kinase is present at a concentration of 100 nM and phosphorylated LCK kinase is present at a concentration of 50 nM. The assay is performed at 37 ° C. under initial rate conditions, and the rate is corrected for the background rate measured in the absence of enzyme. The% inhibition is calculated relative to the control enzyme assayed in the presence of 2% (v / v) DMSO.

General Kinase Inhibition Assay Inhibition of various kinases was monitored by examining the transfer to phosphate to the appropriate peptide substrate for each kinase.

  The activity of Jnk1, Jnk2, Jnk3, IKK1, IKK2EE, p38α, p38β, MKK3, MKK4, MKK4, MKK6, MKK7, cdk2 / E, cdk2 / A, PKCα, ERK, and PKA is SP ) To protein substrate and the product was monitored by precipitation with trichloroacetic acid. ATP was 3 times the Km for the kinase. Activity for Akt1, Akt2 and SGK was monitored by transfer of radiolabeled phosphate from ATP to specific substrate peptide and capture of the peptide on P81 packed filter paper. ATP was the Km for the kinase. The activity of IRTK, AbI and SRC was monitored by transfer of phosphate from ATP to a biotinylated peptide substrate and detection of phosphorylated peptides using the LANCE technology (Perkin Elmer). ATP was 3 times the Km for the kinase.

  Drugs that target two or more different kinases are useful in the methods of the invention described herein. The compounds can be identified and their effects on specific kinases can be identified using the assay briefly described in Examples 4-83 below. Each of these kinases is described in Davies et al., Biochem. J. et al. 351: 96-105 (2000). Unless otherwise noted, all assays were performed with 10 μM ATP.

All kinase-diluted kinases are pre-diluted to 10 × working concentration before being added to the assay. The composition of the dilution buffer for each kinase is described in detail below.

Further, the following abbreviations are used. h is human, r is rat, m is mouse, b is cow, and y is yeast.

Dissolve and dilute all substrates except the substrate histones H1 and AFT2 in a working stock in deionized water. Histone H1 is diluted to 10 × working stock in 20 mM MOPS (pH 7.4) before being added to the assay. AFT2 is typically 20 × in 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1 mM EGTA, 0.03% Brij-35, 50% glyceryl, 1 mM benzamidine, 0.2 mM PMSF and 0.1% β-mercaptoethanol. Save in working stock.

SGk (h) assay In a final reaction volume of 25 μl, SGK (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM GRPRTSFAEGKK (SEQ ID NO: 2), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

GSK3β (h) assay With a final reaction volume of 25 μl, GSK3β (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 20 μM YRRAAVPPSPSLSRHSSPQS (p) EDEEEE (phospho GS2 peptide; SEQ ID NO: 3) Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 50 mM phosphoric acid and once with methanol, then dried and scintillation counted.

AMPK (r) assay In a final reaction volume of 25 μl, AMPK (r) (5-10 mU) was added to 50 mM Hepes (pH 7.4), 1 mM DTT, 0.02% Brij-35, 200 μM AMP, 200 μM AMARAASAALARARRR (SEQ ID NO: 4). ) Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CHK1 (h) assay In a final reaction volume of 25 μl, CHK1 (h) (5-10 mU) was replaced with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 200 μM KKKVSRSLYRSPSMPENLNRPR (SEQ ID NO: 5), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CHK2 (h) Assay In a final reaction volume of 25 μl, CHK2 (h) (5-10 mU) was added 20 mM Hepes (pH 7.6), 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton X- Incubate with 100, 165 μM RRRDDDSDDDDD (SEQ ID NO: 6), 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Lck (h) assay In a final reaction volume of 25 μl, Lck (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 250 μM KVEKIGEGGTYGVVYK (Cdc2 peptide; SEQ ID NO: 7) Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK2 / cyclin A (h) assay In a final reaction volume of 25 μl, CDK2 / cyclin A (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone HCl, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

MAPK2 (m) assay In a final reaction volume of 25 μl, MAPK2 (m) (5-10 mU) is added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

SAPK2a (h) assay In a final reaction volume of 25 μl, SAPK2a (h) (5-10 mU) was added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

SAPK2b (h) assay In a final reaction volume of 25 μl, SAPK2b (h) (5-10 mU) was added 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

SAPK3 (h) assay In a final reaction volume of 25 μl, SAPK3 (h) (5-10 mU) was added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

SAPK4 (h) assay In a final reaction volume of 25 μl, SAPK4 (h) (5-10 mU) was added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

MSK1 (h) assay In a final reaction volume of 25 μl, MSK1 (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM GRPRTSFAEGKK (SEQ ID NO: 2), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKBα (h) assay In a final reaction volume of 25 μl, PKBα (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM GRPRTSFAEGKK (SEQ ID NO: 2), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

ROCK-II (r) assay With a final reaction volume of 25 μl, ROCK-II (r) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 30 μM KEAKEKRRQRIA LSSLSLSKSKSGGSQK (SEQ ID NO: 8), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

P70S6K (h) assay In a final reaction volume of 25 μl, P70S6K (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 100 μM KKRNRTLV (SEQ ID NO: 9), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKA (b) assay In a final reaction volume of 25 μl, PKA (b) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM LRRASLG (Chemptide; SEQ ID NO: 10), 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times with 50 mM phosphoric acid for 5 minutes, washed once with methanol, dried, and scintillated.

MAPKAP-K2 (h) assay With a final reaction volume of 25 μl, MAPKAP-K2 (h) (5-10 mU) is added to 50 mM β-glycerophosphate Na (pH 7.5), 0.1 mM EGTA, 30 μM KKLNRTLSVA (SEQ ID NO: 11). Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

JNK1α1 (h) assay In a final reaction volume of 25 μl, JNK1α1 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 3 μM ATF2, 10 mM Mg acetate. And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

JNK2α2 (h) assay In a final reaction volume of 25 μl, JNK2α2 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 3 μM ATF2, 10 mM Mg acetate. And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

JNK3 (r) assay In a final reaction volume of 25 μl, JNK3 (r) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 250 μM peptide, 10 mM Mg acetate. And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PRAK (h) assay In a final reaction volume of 25 μl, PRAK (h) (5-10 mU) was added to 50 mM β-glycerophosphate Na (pH 7.5), 0.1 mM EGTA, 30 μM KKLRRTLSVA (SEQ ID NO: 11), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 50 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CHK2 (h) assay In a final reaction volume of 25 μl, CHK2 (h) (5-10 mU) was replaced with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 200 μM KKKVSRSLYRSPSMPENLNRPR (SEQ ID NO: 5), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

MAPK1 (h) assay In a final reaction volume of 25 μl, MAPK1 (h) (5-10 mU) is added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 200 μM peptide, 10 mM Mg acetate and [γ- ³³ P-ATP]. Incubate with (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

c-RAF (h) assay In a final reaction volume of 25 μl, c-RAF (h) (5-10 mU) is added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.66 mg / ml myelin basic protein, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK1 / cyclin B (h) assay In a final reaction volume of 25 μl, CDK1 / cyclin B (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

cSRC (h) assay In a final reaction volume of 25 μl, cSRC (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 250 μM KVEKIGETGTYGVVYK (Cdc2 peptide; SEQ ID NO: 7), 10 mM Mg acetate and Incubate with [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CaMKII (r) assay In a final reaction volume of 25 μl, CaMKII (r) (5-10 mU) was added to 40 mM Hepes (pH 7.4), 5 mM CaCl ₂ , 30 μg / ml calmodulin, 30 μM KKLNRTLSVA (SEQ ID NO: 11), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PRK2 (h) assay In a final reaction volume of 25 μl, PRK2 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 30 μM AKRRRLSSSLRA (SEQ ID NO: 12 ) Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PDK1 (h) assay In a final reaction volume of 25 μl, PDK1 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 100 μM KTFCGTPEYLAPEVRREPRISEEEQEMFRDFDYDYADWC (SEQ ID NO: 13) (PDKtide), 0.1% β-mercaptoethanol, Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Fyn (h) assay In a final reaction volume of 25 μl, Fyn (h) (5-10 mU) was converted to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 250 μM KVEKIGEEGTYCVVVK (Cdc2 peptide; SEQ ID NO: 7) Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKCα (h) assay PKCα (h) (5-10 mU) in 20 mM Hepes (pH 7.4), 0.03% Triton X-100, 0.1 mM CaCl ₂ , 0.1 mg / ml in a final reaction volume of 25 μl. Incubate with phosphatidylserine, 10 μg / ml diacylglycerol, 0.1 mg / ml histone H1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKCβII (h) assay In a final reaction volume of 25 μl, PKCβII (h) (5-10 mU) was added to 20 mM Hepes (pH 7.4), 0.03% Triton X-100, 0.1 mM CaCl ₂ , 0.1 mg / ml. Incubate with phosphatidylserine, 10 μg / ml diacylglycerol, 0.1 mg / ml histone H1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKCγ (h) Assay PKCγ (h) (5-10 mU) in 20 mM Hepes (pH 7.4), 0.03% Triton X-100, 0.1 mM CaCl ₂ , 0.1 mg / ml in a final reaction volume of 25 μl. Incubate with phosphatidylserine, 10 μg / ml diacylglycerol, 0.1 mg / ml histone H1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CK1 (y) assay In a final reaction volume of 25 μl, CK1 (y) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 250 μM KRRRALS (p) VASLPGL (SEQ ID NO: 14), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

ZAP-70 (h) assay With a final reaction volume of 25 μl, ZAP-70 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol, 0.1 mg / ml poly (Glu, Tyr) 4: ₁ , 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration) Incubate with. The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

MEK1 (h) assay In a final reaction volume of 25 μl, MEK1 (h) (1-5 mU) was added to 50 mM Tris (pH 7.5), 0.2 mM EGTA, 0.1% β-mercaptoethanol, 0.01% Brij- 35. Incubate with 1 μM inactive MAPK2 (m), 10 mM Mg acetate and cold ATP (ratio required concentration). The reaction is started by adding MgATP. After 40 minutes incubation at room temperature, the incubation mixture (5 μl) is used to p. 7. Start the MAPK2 (m) assay described in 7.

MKK4 (m) assay In a final reaction volume of 25 μl, MKK4 (m) (1-5 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.1 mM Na valerate. Incubate with 2 μM inert JNK1α1 (h), 10 mM Mg acetate and cold ATP (required concentration). The reaction is started by adding MgATP. After incubating at room temperature for 40 minutes, this incubation mixture (5 μl) was used to p.p. in this book except that AFT2 was replaced with 250 μM peptide. The JNK1α1 (h) assay, exactly described in 10, is initiated.

MKK7β (h) assay In a final reaction volume of 25 μl, MKK7β (h) (1-5 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.1 mM Na valerate. Incubate with 2 μM inert JNK1α1 (h), 10 mM Mg acetate and cold ATP (required concentration). The reaction is started by adding MgATP. After incubating at room temperature for 40 minutes, this incubation mixture (5 μl) was used to p.p. in this book except that AFT2 was replaced with 250 μM peptide. The JNK1α1 (h) assay, exactly described in 10, is initiated.

MKK6 (h) assay In a final reaction volume of 25 μl, MKK6 (h) (1-5 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.1 mM Na valerate. Incubate with 1 mg / ml BSA, 1 μM inactive SAPK2a (h), 10 mM Mg acetate and cold ATP (required concentration). The reaction is started by adding MgATP. After 40 minutes incubation at room temperature, the incubation mixture (5 μl) is used to p. The SAPK2a (h) assay described in 8 is started.

IKKα (h) assay In a final reaction volume of 25 μl, IKKα (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 200 μM peptide, 10 mM Mg acetate and [γ- ³³ P-ATP]. Incubate with (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

IKKβ (h) assay In a final reaction volume of 25 μl, IKKβ (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 100 μM peptide, 10 mM Mg acetate and [γ- ³³ P-ATP]. Incubate with (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKCθ (h) assay In a final reaction volume of 5 μl, PKCθ (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CaMKIV (h) assay In a final reaction volume of 25 μl, CaMKIV (h) (5-10 mU) was converted to 40 mM Hepes (pH 7.4), 5 mM CaCl ₂ , 30 μg / ml calmodulin, 30 μM KKLNRTLSVA (SEQ ID NO: 11), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Blk (m) assay In a final reaction volume of 25 μl, Blk (m) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. Incubate with 0.1 mg / ml poly (Glu, Tyr) 4: 1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Syk (h) assay In a final reaction volume of 25 μl, Syk (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. Incubate with 0.1 mg / ml poly (Glu, Tyr) 4: 1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CSK (h) assay In a final reaction volume of 25 μl, CSK (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. , 0.1 mg / ml poly (Glu, Tyr) 4: ₁ , 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Lyn (m) assay In a final reaction volume of 25 μl, Lyn (m) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. Incubate with 0.1 mg / ml poly (Glu, Tyr) 4: 1, 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK3 / cyclin E (h) assay In a final reaction volume of 25 μl, CDK3 / cyclin E (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK5 / p35 (h) assay In a final reaction volume of 25 μl, CDK5 / p35 (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM Mg acetate. And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK2 / cyclin E (h) assay In a final reaction volume of 25 μl, CDK2 / cyclin E (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK6 / cyclin D3 (h) assay In a final reaction volume of 25 μl, CDK6 / cyclin D3 (h) (5-10 mU) is converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml histone H1, 10 mM. Incubate with Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

CDK7 / cyclin H / MAT1 (h) assay In a final reaction volume of 25 μl, CDK7 / cyclin H / MAT1 (h) (5-10 mU) is converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 500 μM peptide, 10 mM acetic acid. Incubate with Mg and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Rsk3 (h) assay In a final reaction volume of 25 μl, Rsk3 (h) (5-10 mU) was replaced with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM KKKKNLTLSVA (SEQ ID NO: 11), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

IR (h) assay In a final reaction volume of 25 μl, IR (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. , 250 μM KKSRGDYMTMQIG (SEQ ID NO: 15), 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

IGF-1R (h) assay In a final reaction volume of 25 μl, IGF-1R (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% Incubate with β-mercaptoethanol, 250 μM KKKSPGEYVNIEFG (SEQ ID NO: 16), 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKBβ (h) assay In a final reaction volume of 25 μl, PKBβ (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM GRPRTSFAEGKK (SEQ ID NO: 2), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

FGFR3 (h) assay In a final reaction volume of 25 μl, FGFR3 (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM Na valerate, 0.1% β-mercaptoethanol. , 0.1 mg / ml poly (Glu, Tyr) 4: ₁ , 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PDGFRα (h) assay In a final reaction volume of 25 μl, PDGFRα (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml poly (Glu, Tyr) 4: 1, Incubate with 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PDGFRβ (h) assay In a final reaction volume of 25 μl, PDGFRβ (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml poly (Glu, Tyr) 4: 1, Incubate with 10 mM MnCl ₂ , 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

MAPK2 (h) assay In a final reaction volume of 25 μl, MAPK2 (h) (5-10 mU) was added to 25 mM Tris (pH 7.5), 0.02 mM EGTA, 0.33 mg / ml myelin basic protein, 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

ROCK-II (h) assay In a final reaction volume of 25 μl, ROCK-II (h) (5-10 mU) was added to 50 mM Tris (pH 7.5), 0.1 mM EGTA, 30 μM KEAKEKRRQRIA LSSLSLSKSKSGGSQK (SEQ ID NO: 8), 10 mM Mg acetate And [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKA (h) assay In a final reaction volume of 25 μl, PKA (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM LRRASLG (Chemptide; SEQ ID NO: 10), 10 mM Mg acetate and [ γ- ³³ P-ATP] incubated with (specific activity approximately 500 cpm / pmol, concentration as required). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 50 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Rsk1 (r) assay In a final reaction volume of 25 μl, Rsk1 (r) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM KKKKNLTLSVA (SEQ ID NO: 11), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Rsk2 (h) assay In a final reaction volume of 25 μl, Rsk2 (h) (5-10 mU) was replaced with 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM KKKKNLTLSVA (SEQ ID NO: 11), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PAK2 (h) assay In a final reaction volume of 25 μl, PAK2 (h) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 30 μM KEAKEKRRQEIAKRRRRLSSLSLASTSKSGGSQK (SEQ ID NO: 8), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Fes (h) assay In a final reaction volume of 25 μl, Fes (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml poly (Glu, Tyr) 4: 1, Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Yes (h) assay In a final reaction volume of 25 μl, Yes (h) (5-10 mU) was converted to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1 mg / ml poly (Glu, Tyr) 4: 1, Incubate with 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on filter mat A, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

ABL (m) assay In a final reaction volume of 25 μl, ABL (m) (5-10 mU) was added to 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 50 μM EAIYAAPFAKKKK (SEQ ID NO: 17), 10 mM Mg acetate and [γ- ^[ 33P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

PKCε (h) assay In a final reaction volume of 25 μl, PKCε (h) (5-10 mU) was added to 20 mM Hepes (pH 7.4), 0.03% Triton X-100, 0.1 mg / ml phosphatidylserine, 10 μg / ml. Incubate with diacylglycerol, 50 μM ERMRPRKRQGSVRRRV (SEQ ID NO: 18), 10 mM Mg acetate and [γ- ³³ P-ATP] (specific activity about 500 cpm / pmol, required concentration). The reaction is started by adding Mg ²⁺ [γ- ³³ P-ATP]. After 40 minutes incubation at room temperature, the reaction is stopped by adding 3% phosphoric acid solution (5 μl). The reaction (10 μl) is then spotted on a P30 filter mat, washed 3 times for 5 minutes with 75 mM phosphoric acid and once with methanol, then dried and scintillation counted.

Synthesis of 3- (6-methoxynaphthalen-2-yl) -5- (5-morpholin-4-ylmethyl-1H- [1,2,4] triazol-3-yl) -1H-indazole (CC001)

A. Morpholin-4-yl-acetic acid hydrazide A solution of morpholine methyl acetate (2.43 g, 15.3 mmol), ethanol (20 ml) and hydrazine (0.53 ml, 16.8 mmol) was stirred at 90 ° C. for 18 hours. The mixture was concentrated and dried to give the title compound (2.30 g, 95%). ES-MS (m / z) = 160 [M + l] ^< +>.

B. 3- (6-Methoxynaphthalen-2-yl) -5- (5-morpholin-4-ylmethyl-1H- [1,2,4] triazol-3-yl) -1H-indazole The title compound is Manufactured. The flask was charged with 3- (6-methoxynaphthalen-2-yl) -1H-indazole-5-carboxymido acid ethyl ester (1.0 g, 2.62 mmol) and morpholin-4-yl-acetic acid hydrazide (1.67 g, 10.5 mmol). After 10 minutes, hydrazide prepared as described in Example 422A of WO 02/10137 (1.05 g, 7.31 mmol) was added and the mixture was heated at 90 ° C. for 18 hours. . The mixture was concentrated and purified by preparative HPLC to give the title compound (481 mg, 42%). ¹ H NMR (CD ₃ OD) δ 8.82 (s, 1H), 8.42 (s, 1H), 8.08 (d, 2H), 7.93 (t, 2H), 7.65 (br s, 1H), 7.30 (s, 1H), 7.21 (d, 1H), 4.15 (s, 3H), 3.72 (m, 6H), 2.59 (brs, 4H) ES-MS (m / z) = 441 [M + 1] ⁺ .

5- (5-Isobutyl-1H- [1,2,4] triazol-3-yl) -3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole Synthesis of (CC002)

A. 4-Fluoro-3- [hydroxy- (6-methoxy-naphthalen-2-yl) -methyl] benzonitrile LDA (24.5 ml, 49 mmol) in a cold (−78 ° C.) solution in THF (40 ml). 4-Fluorobenzonitrile (5.45 g, 45 mmol) in THF (30 ml) and 6-methoxy-2-naphthaldehyde (9.13 g, 49 mmol) in THF (40 ml) were added. The reaction was stirred at −78 ° C. for 1 hour and then warmed to room temperature over 2 hours. The reaction was quenched with ice and THF was removed in vacuo. The aqueous solution was then extracted with ethyl acetate. The organic phase was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude material was purified by column chromatography _(SiO 2, 4: 1 hexane: ethyl acetate) to give the title compound (5.5 g, 40% yield). ES-MS (m / z) = 308 [M + 1] ^< +>.

B. 4-Fluoro-3- (6-methoxynaphthalene-2-carbonyl) -benzonitrile Pyridinium chlorochromate (6.4 g, 29.7 mmol) was absorbed into a slurry of dichloromethane (40 ml) in a round bottom flask. 4-Fluoro-3- [hydroxy- (6-methoxy-naphthalen-2-yl) -methyl] benzonitrile (6.08 g, 19.8 mmol) in dichloromethane (40 ml) was added. The reaction became black. The mixture was stirred at room temperature for 5 hours, then filtered through a celite pad and the solvent removed in vacuo. The crude material was purified by column chromatography _(SiO 2, 4: 1 hexane: ethyl acetate -1: 1 hexanes: ethyl acetate) to give the title compound (4.54 g, 75% yield). ES-MS (m / z) = 306 [M + l] ^< +>.

C. 4-Fluoro-3- (6-hydroxynaphthalene-2-carbonyl) -benzonitrile 4-Fluoro-3- (6-methoxynaphthalene-2-carbonyl) -benzonitrile (3.976 g, 13 mmol) was added to dichloromethane (350 ml). ) And cooled in an ice bath under nitrogen. Boron tribromide (12.28 ml, 130 mmol) was added slowly and the reaction was stirred at room temperature overnight. The reaction was quenched with ice, neutralized with sodium bicarbonate and extracted with dichloromethane. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo. The crude material was purified by column chromatography _(SiO 2, 7: 3 hexane: ethyl acetate) to give the title compound (2.5 g, 66% yield). ES-MS (m / z) = 292 [M + l] ^< +>.

D. 4-fluoro-3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalene-2-carbonyl] -benzonitrile 4-fluoro-3- (6-hydroxynaphthalene-2-carbonyl) -benzonitrile ( 2.7 g, 9.3 mmol) was dissolved in dioxane (22 ml). Tetraethylammonium bromide (195 mg, 0.93 mmol) and sodium hydroxide (1.12 g in 16 ml water) were added sequentially. 1- (2-Chloroethyl) pyrrolidine hydrochloride (1.74 g, 10.23 mmol) was added and the reaction mixture was stirred at 55 ° C. for 4 hours. The solvent was removed in vacuo and water was added. The reaction mixture was neutralized to pH 7 and extracted thoroughly with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and the solvent removed in vacuo to give the title compound (3.16 g, 87% yield). ES-MS (m / z) = 389 [M + l] ^< +>.

E. 3- [6- (2-Pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole-5-carbonitrile 4-fluoro-3- [6- (2-pyrrolidin-1-yl-) Hydrazine monohydrate (0.87 ml, 17.9 mmol) was added to a solution of (ethoxy) -naphthalene-2-carbonyl] -benzonitrile (3.16 g, 8.14 mmol) in toluene (40 ml). The reaction mixture was heated at 65 ° C. overnight. The solvent was removed in vacuo and the crude material was purified by column chromatography _(SiO 2, 1% triethylamine / ethyl acetate) to give the title compound (2.0 g, 65% yield). ES-MS (m / z) = 383 [M + 1] ^< +>.

F. 3- [6- (2-Pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole-5-carboxymido acid ethyl ester hydrochloride The title compound was prepared as follows. A solution of 3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole-5-carbonitrile (2 g, 5.23 mmol) in ethanol (400 ml). Cooled to 0 ° C. HCl gas was passed through the reaction mixture for 30 minutes. The reaction vessel was sealed and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with diethyl ether and the precipitate was filtered and washed with diethyl ether. The white solid was dried in vacuo at 40 ° C. overnight to give the title compound as a yellow solid (2 g, 89% yield). ES-MS (m / z) = 429 [M + 1] ^< +>.

G. 5- (5-Isobutyl-1H- [1,2,4] triazol-3-yl) -3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole The title compound was prepared as follows. 3-Methylbutyric acid in a solution of 3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole-5-carboxymido acid ethyl ester (2 g, 4.67 mmol) Hydrazide (1.63 g, 14.04 mmol) and triethylamine (13.04 ml, 93.44 mmol) in methanol (20 ml) were added. The reaction was stirred in a sealed pressure vessel at 100 ° C. for 4 hours. Concentrated and purified by HPLC (20-65% water / acetonitrile) to give the title compound (378.5 mg, 17% yield). ¹ H NMR (methanol-d ₄ , 300 MHz) δ 8.82 (s, 1H), 8.47 (s, 1H), 8.10 (dd, 2H), 7.97 (dd.2H), 7. 69 (d, 1H), 7.39 (s, 1H), 7.29 (d, 1H), 4.47 (t, 2H), 3.72 (m, 4H), 3.28 (m, 2H) ), 2.75 (d, 2H), 2.07 (m, 5H), 1.01 (d, 6H); ES-MS (m / z) = 481 [M + 1] ⁺ .

5- [5- (2,2-Dimethylpropyl) -1H- [1,2,4] triazol-3-yl] -3- [6- (pyridin-2-ylmethoxy) -naphthalen-2-yl]- Synthesis of 1H-indazole (CC004)

A. 2- (6-Bromo-naphthalen-2-yloxymethyl) -pyridine 6-bromonaphthol (6.13 g, 27.5 mmol), pyridin-2-yl-methanol (2.64 ml, 27.5 mmol, 1 0.0eq) and triphenylphosphine (10.8 g, 41.30 mmol, 1.5 eq) in THF were cooled in an ice bath whereupon diisopropyl azodicarboxylate (8.12 ml, 41.3 mmol, 1.5 eq) was added. The reaction was monitored by TLC (30% ethyl acetate / hexane) and was complete after 24 hours. The solvent was removed in vacuo and subjected to Biotage column chromatography to give the title compound (9.00 g, 100% yield) as a brown solid. ES-MS (m / z) = 313 [M + 1].

B. 3- [6- (Pyridin-2-ylmethoxy) naphthalen-2-yl] -1- (tetrahydro-pyran-2-yl) -1H-indazole-5-carbonitrile 2- (6-Bromo-naphthalene-2- In a solution of yloxymethyl) -pyridine (4.97 g, 15.8 mmol) in DMF (50 ml), bis (pinnacarato) diboron (4.02 g, 15.8 mmol, 1.0 eq), potassium acetate (4 .66 g. 47.6 mmol, 3.0 eq) and palladium chloride II (bis-diphenylphosphinoferrocene) dichloromethane (1.29 g, 10% mmol) were added. The reaction was heated at 80 ° C. overnight and LCMS confirmed the formation of boronate ester complex. To the reaction was added 3-bromo-1-perhydro-2H-pyran-2-yl-1H-indazole-5-carbonitrile (4.85 g, 15.8 mmol, 1.0 eq) and potassium phosphate (10.09 g, 47.6 mmol.3.0 eq) was added and stirred at 80 ° C. overnight. The reaction was monitored by LCMS and was complete after 24 hours. The solvent was removed in vacuo and the residue was washed with water, extracted with ethyl acetate and subjected to Biotage column chromatography (60% ethyl acetate / hexane) to give the title compound (2.10 g, 30% yield) as a brown solid Got as. ES-MS (m / z) = 460 [M + 1].

C. 3- [6- (Pyridin-2-ylmethoxy) -naphthalen-2-yl] -1H-indazole-5-carboxymido acid ethyl ester 3- [6- (Pyridin-2-ylmethoxy) naphthalen-2-yl]- A solution of 1- (tetrahydro-pyran-2-yl) -1H-indazole-5-carbonitrile (2.00 g, 4.30 mmol) in ethanol (500 ml) was cooled in a dry ice / acetone bath, HCl (g) was passed through for 20 minutes. The reaction was monitored by LCMS and was complete after 72 hours. The solvent was removed in vacuo and triturated with diethyl ether. The solid was filtered to give the title compound (1.05 g, 58% yield) as an off-white solid. ES-MS (m / z) = 422 [M + 1].

D. 5- [5- (2,2-Dimethylpropyl) -1H- [1,2,4] triazol-3-yl] -3- [6- (pyridin-2-ylmethoxy) -naphthalen-2-yl]- 1H-indazole 3- [6- (Pyridin-2-ylmethoxy) -naphthalen-2-yl] -1H-indazole-5-carboxymido acid ethyl ester (0.50 g, 1.18 mmol) was added to a solution containing dimethylbutyric acid. Hydrazide (0.62 g, 4.72 mmol, 4.0 eq) and triethylamine (3.28 ml, 23.6 mmol, 20.0 eq) were added. The reaction was heated to 90 ° C. overnight in a sealed tube. The reaction was monitored by LCMS and was complete after 24 hours. The solvent was removed in vacuo and subjected to preparative HPLC (20-80% acetonitrile / water + 0.1% TFA) to give the title compound (60 mg, 9% yield) as a white solid. ¹ H NMR (CD ₃ OD) δ 8.98 (s, 1H), 8.70 (d, 1H), 8.60 (s, 1H), 8.35 (dd, 2H), 8.10 (d , 1H), 8.00 (dd, 2H), 7.80 (m, 2H), 7.55-7.40 (m, 3H), 5.45 (s, 2H), 2.95 (s, 2H), 1.05 (s, 9H); ES-MS (m / z) = 566 [M + 1].

2-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] -6- {5- [3- (2,2-dimethylpropyl) (1H-1,2,4-triazol-5-yl )] Synthesis of (1H-indazol-3-yl)} naphthalene (CC005)

A. 2-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] -6-bromonaphthalene The title compound was prepared as follows. 6-Bromo-2-naphthol (6.95 g, 31.2 mmol), ((2S) -1-ethylpyrrolidin-2-yl) methane-1-ol (6.20 g, 48.13 mmol) and triphenyl To a solution of phosphine (12.26 g, 46.8 mmol) in THF was added diisobutyl azodicarboxylate (9.23 ml, 46.8 mmol). The solution was stirred at ambient temperature for 20 minutes and monitored by TLC until the reaction was complete. The solvent was evaporated under reduced pressure to yield an oil. To this oil was added a mixture of diethyl ether: hexane (1: 1) and sonicated for 5 minutes to precipitate triphenylphosphine oxide. The white solid was filtered through celite and the resulting filtrate was condensed to an oil under reduced pressure. The oil was purified by silica gel chromatography (3-10% methanol / dichloromethane) to give the title compound (11.0 g, yield> 100%). ES-MS (m / z) = 334 [M + 1] ⁺ , 336 [M + 2] ⁺ .

B. 3- {6-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] (2-naphthyl)}-1-perhydro-2H-pyran-2-yl-1H-indazole-5-carbonitrile The compound was prepared as follows. 2-[((2S) -1-Ethylpyrrolidin-2-yl) methoxy] -6-bromonaphthalene (4.46 g, 13.39 mmol) in dimethylformamide (70 ml) with [1,1 ′ -Bis (diphenylphosphinoferrocene)] complex (1: 1) (1.13 g, 1.385 mmol), potassium acetate (4.07 g, 41.55 mmol) and bis (piminacholato) diborane (3.51 g, 13.85 mmol) of the mixture was heated to 95 ° C. for 3 hours. The reaction was monitored by TLC and ES-MS to confirm the formation of boronate ester. 3-Bromo-1- (tetrahydro-pyran-2-yl) -1H-indazole-5-carbonitrile (4.23 g, 13.85 mmol) and potassium carbonate (10.85 g, 41.55 mmol) were then added. Add and heat at 95 ° C. for an additional 16 hours. The resulting mixture was then condensed under reduced pressure to yield a black oil. The oil was then diluted with ethyl acetate, filtered through celite and the solvent removed under reduced pressure. The resulting oil was purified by silica gel chromatography (10-15% methanol / dichloromethane) to give the title compound (1.05 g, 16% yield). ES-MS (m / z) = 481 [M + 1] ⁺ .

C. (3- {6-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] (2-naphthyl)} (1H-indazol-5-yl)) ethoxymethanimine The title compound was prepared as follows: did. 3- {6-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] (2-naphthyl)}-1-perhydro-2H-pyran-2-yl-1H-indazole-5-carbonitrile ( 4.6 g, 8.85 mmol) was dissolved in ethanol (800 ml) and cooled to 0 ° C. Hydrogen chloride gas was then passed through the solution for 20 minutes. The acidified mixture was then stirred at room temperature for 16 hours. The resulting solution was condensed under reduced pressure to yield a solid. This solid was washed with diethyl ether, filtered through a Buchner funnel and dried under vacuum to give the title compound (1.10 g, 98% yield). ES-MS (m / z) = 443 [M + 1] ^< +>.

D. [((2S) -1-ethylpyrrolidin-2-yl) methoxy] -6- {5- [3- (2,2-dimethylpropyl) (1H-1,2,4-triazol-5-yl)] (1H-Indazol-3-yl)} naphthalene The title compound was prepared as follows. (3- {6-[((2S) -1-ethylpyrrolidin-2-yl) methoxy] (2-naphthyl)} (1H-indazol-5-yl)) ethoxymethanimine (0.550 g, 1.24 Mmol) and N-amino-3,3-dimethylbutanamide (0.323 g, 2.488 mmol) in a solution containing cyclopentylmethyl hydrazide (1.4 g, 9.89 mmol) and triethylamine (4.98 g, 49.49 mmol). 4 mmol) was added. The mixture was heated in a sealed tube to 95 ° C. for 16 hours. The solvent was then removed under reduced pressure and the oil was purified by preparative HPLC (20-80% acetonitrile / water, 60 ml / min) to give the title compound (0.061 g, 9.6% yield). It was. This compound was 100% pure by HPLC analysis. . ¹ H NMR (CHCl ₃ ) δ 8.83 (s, 1H), 8.39 (s, 1H), 8.20 (d, 1H), 8.09 (d, 1H), 7.83 (m, 2H), 7.58 (d, 1H), 7.19 (m, 2H), 4.16 (m, 1H), 4.0 (m, 1H), 3.26 (t, 1H), 3. 05 (m, 1H), 2.97 (m, 1H), 2.78 (s, 2H), 2.50 (m, 1H), 2.33 (m, 1H), 2.21 (m, 1H) ), 2.05 (m, 1H), 1.85 (m, 4H), 1.10 (t, 3H), 1.04 (q, 2H); ES-MS (m / z) = 509 [M + 1] ] ⁺ .

  Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be encompassed by the claims.

  All publications and patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication and patent document was specifically incorporated by reference herein. Incorporate.

The results of CC001 administration versus tumor size in SCID mouse xenograft model given HCT-116 cells are shown. The results of CC002 or CC003 administration on tumor size in the SCID mouse xenograft model given HCT-116 cells are shown. The results of CC001 or CC001 and camptosa administration on tumor size in SCID mouse xenograft model given HCT-116 cells are shown.

Claims (15)

  Administering to an individual a single drug that modulates the activity of at least two kinases, each of said kinases from CDK kinase, Rsk kinase, MAPK kinase, checkpoint kinase, Src kinase, Fes, Lyn, Syk A method for treating said individual selected independently from the group consisting of:   At least two kinases are cSRC, Yes, Fyn, Lck, Fes, Lyn, Syk, Rsk1, CDK1, CDK2, CDK3, CDK5, CDK6, CDK7, CHK1, CHK2, JNK1, MAPK1, MAPK2, MAPKAP-K5, MEK1, The method according to claim 1, wherein there are at least two of ROCKII, PRK2, PRAK, p70S6 or Aurora A.   The method of claim 2, wherein the at least two kinases are human CDK1, CDK2, cSRC, Yes, MEK1 and Rsk1.   The method according to claim 1 or 2, wherein the individual suffers from cancer, proliferative disease, inflammatory disease or obesity. Single drug structure:

[Where:
R ¹ is —H, —halo, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , — ^{CO (R) 5, -OCO (} R) 5, -NHC (O) R 5, -SO 2 R 6, - aryl, - heterocycle, - heteroaryl, - cycloalkyl, - _{(C 1-6} alkylene) -R ² or -O- (C _1-6 alkylene) -R ² ;
R ² represents —H, —halo, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , — ^{C (O) R 5, -OC} (O) R 5, -NHC (O) R 5, -SO 2 R 6, - aryl, - heterocyclic, - cycloalkyl - heteroaryl or;
R ³ is independently -H, -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, -C _1-6 haloalkyl, -cycloalkyl, -aryl or -heterocycle;
Each R ⁴ is independently —H, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —cycloalkyl, —aryl, —heterocycle or — (C _1-6 alkylene); -OR ³ ;
R ⁵ is —H, —C _1-6 alkyl, —C _1-6 alkenyl, —C _1-6 alkynyl, —cycloalkyl, —aryl, —heterocycle, —OR ³ or —N (R ⁴ ) ₂ . Yes;
R ⁶ is -H, -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, -N (R ⁴ ) ₂ , -cycloalkyl, -aryl or -heterocycle, -C (R < ⁷ >)-or -N- (wherein up to three Z can be -N-);
R ⁷ is —H, —halo, —C _1-6 alkyl, —C _1-6 haloalkyl, —O— (C _1-6 haloalkyl), —C _1-6 alkenyl, —C _1-6 alkynyl, —OR ³ , —N (R ⁴ ) ₂ , —CN, —NO ₂ , —C (O) R ⁵ , —OC (O) R ⁵ , —NHC (O) R ⁵ , —SO ₂ R ⁶ , —aryl, - heterocycle, - cycloalkyl, -C (O) NH- _{(C 1-6 alkylene) n} - cycloalkyl, -C (O) NH- _{(C 1-6 alkylene) n} - aryl, -C (O) NH- (C _1-6 alkylene) _n -heterocycle,-(C _1-6 alkylene) cycloalkyl,-(C _1-6 alkylene) -aryl,-(C _1-6 alkylene) -heterocycle, -O - _{(C 1-6} ^{alkylene) -C (O) R 5,} -O- (C 1-6 alkylene) —N (R ⁴ ) ₂ , —O (C _1-6 alkylene) -cycloalkyl, —O— (C _1-6 alkylene) -aryl or —O— (C _1-6 alkylene) -heterocycle, wherein , R ⁷ is attached to the bicyclic ring system via a carbon atom, and n is 0 or 1);
R ⁹ is —H, —C _1-6 alkyl or cycloalkyl]
And the isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrates thereof. A single drug has the structure:

[Where:
R ¹ is —H, —C _1-6 alkyl, — (C _1-6 alkylene) -R ² or —O— (C _1-6 alkylene) -R ² ;
R ² is —C _1-6 alkyl, —C _1-6 alkoxy, —OH, —N (R ³ ) ₂ , —aryl, —heteroaryl, —heterocycle or —cycloalkyl;
Each R ³ is independently —H, —C _1-6 alkyl or —C _1-6 alkylene- (C _1-6 alkoxy);
R ⁴ represents —N (R ⁵ ) ₂ , —O—C _1-6 alkyl, —C (O) NH— (C _1-6 alkylene) _m -heterocycle, —C (O) -heterocycle, —C (O) NH- (C _1-6 alkylene) ^m -heteroaryl, -C (O) -heteroaryl,-(C _1-6 alkylene) -cycloalkyl, -O- (C _1-6 alkylene) -N _{^{(R 5) 2, -O- (}} C 1-6 _{alkylene) m} - heterocycle, -O- _{(C 1-6 alkylene) m} - heteroaryl, -O- _{(C 1-6 alkylene) m} - cycloalkyl Or —O— (C _1-6 alkylene) _m —C (O) R ⁵ ;
Z is —CH— or —N—;
Each R ⁵ is independently —H or —C _1-6 alkyl;
m is 0 or 1]
And the isomers, prodrugs, pharmaceutically acceptable salts, solvates and hydrates thereof.   The single drug is 3- (6-methoxynaphthalen-2-yl) -5- (5-morpholin-4-ylmethyl) -1H- [1,2,4] triazol-3-yl) -1H-indazole, 5 -(5-isobutyl-1H- [1,2,4] triazol-3-yl) -3- [6- (2-pyrrolidin-1-yl-ethoxy) -naphthalen-2-yl] -1H-indazole, 5- [5- (2,2-Dimethyl-propyl) -1H- [1,2,4] triazol-3-yl] -3- [6- (pyridin-2-ylmethoxy) -naphthalen-2-yl] -1H-indazole, or 2-[((2s) -1-ethylpyrrolidin-2-yl) methoxy] -6- {5- [3- (2,2-dimethylpropyl) (1H-1,2,4 -Triazol-5-yl)] (1H-indazol-3 The method of claim 1-yl)} naphthalene.   2. The method of claim 1, wherein the single drug is a non-indazole small molecule, peptide or polynucleotide.   A single drug is 5- (5-cyclopentyl-1H- [1,2,4] triazol-3-yl) -3- (4-fluoro-phenyl) -1H-indazole or 3- (5- (1H-1 , 2,4-Triazol-3-yl) (1H-indazol-3-yl)) phenyl-N-cyclopentylcarboxamide is an indazole-containing compound.   When a single drug is present at a concentration of 3 μM, the single drug exhibits the activity of each of the at least 7 kinases shown in Table 2 in the absence of a single drug. 2. The method of claim 1, wherein the method inhibits at least 90% compared to.   When a single drug is present at a concentration of 3 μM, the single drug has the activity of human CDK1, CHK2, PRK2, and ROCK-II in the absence of a single drug, said CDK1, CHK2, The method according to claim 1, which inhibits 90% or more compared to the activity of PRK2 and RPCK-II.   2. The method of claim 1, wherein a single drug is present in the pharmaceutical composition.   The method of claim 1, wherein the individual is treated with a plurality of single drugs.   5. The method of claim 4, wherein the individual is further administered a cancer therapeutic or treatment other than a single drug.   The method of claim 4, wherein the individual is treated for lung cancer or colon cancer.

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